The Aetiology and Pathogenesis
of
Tropical Ulcer
Town
Beverley Adriaans Tropical DermatologyCape Unit Department ofof Medical Microbiology London School of Hygiene and Tropical Medicine
Keppel Str eet
London WClE 7HT
England University
A Thesis presented for the M.D. degree in the
University of Cape Town, South Africa, 1988 .
The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgementTown of the source. The thesis is to be used for private study or non- commercial research purposes only. Cape Published by the University ofof Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author.
University ABSTRACT
Tropical ulcer is a very specific form of cutaneous ulceration. It occurs worldwide in most tropical and a number of subtropical areas.
The disease occurs mainly in older children and young adults with children under the age of 5 and adults over 45 years rarely being affected. Ulcers occur most commonly on the lower leg but may occur on the upper limb. Although most ulcers normally heal slowly over many weeks or months, some ulcers may recur. Recognised complications include squamous cell carcinoma, gangrene and osteitis, although these are rare.
A number of authors have reported on the disease and suggested diet, trauma and infection as aetiological factors for this condition.
This survey was thus conducted to assess as many of these factors as possible. The study took place in 5 tropical areas, namely
Zambia, Gambia, southern India, Fiji and Papua New Guinea.
Consultations took place at hospitals, rural clinics, health centres and villages.
Although many authors have suggested that the disease is related to malnutrition, few have objectively assessed the nutritional status of the patients and compared it with controls. Those studies which included objective assessments were limited to small areas and only investigated specific parameters. In order to investigate the immune response of the host to an anaerobic infection, the antibody levels to the organisms isolated from the ulcers were measured by an ELISA test . The local host response to an infection with a Fusobacterium species was assessed by the number of antibody secreting B-lymphocytes at the site of the ulcers. These parameters ma y play a role in the localisation of the ulcers and account for recurrent infections . In this survey the nutritional status of the patients was objectively assessed and compared to a control population in each area. A questionnaire was used to obtain personal details and information about the present and/ or previous ulcers .
Several authors have also reported fusiform bacilli and spirochaetes on smears from the ulcers. These organisms have not been fully characterized as they are anaerobic bacteria and require a strict anaerobic environment to survive . A suitable transport sys tem wa s therefore developed to transport the specimens from the tropical study areas to London. This kept the samples anaerobic and ensured that the organisms remained viable. During this study, swabs were taken from the base of the ulcers and biopsies from the edge. Both types of samples were examined bacteriologically and all were cultured aerobically and anaerobically .
The biopsies were also examined histologically . Electron microscopy was performed on some biopsies.
Th e isolates were tested for in-vitro cytotoxicity as a possible mechanism for the development of ulcers . The pathogenicity of all the bacterial species recovered from the ulcers was further assessed by their ability either singly, or in combination, to induce an experimental abscess in a guinea pig . In-vivo synergy between the various organisms was also investigated. The guinea pig proved to be the most suitable animal model . Acknowledgements
I would like to thank my two supervisors at the London School of
Hygiene and Tropical Medicine, Dr Roderick J Hay, Reader in
Clinical Mycology and Dr Bohumil S Drasar, Reader in Bacteriology for their help, encouragement and constant advice in conducting this research and Zerin Ahmet, for the technical help she provided throughout the research. Dr Derek Robinson very kindly collected samples and data from some of the areas surveyed. My research was generously supported by the Wellcome Trust. CONT ENT S
Page
SUMMARY l
INTRODUCTION
Study plan 3
REVIEW OF THE LITERATURE 7
Epidemiology 8 Clinical manifestations 16 Nutritional status 25 Role of infection 32 Pathology 41 Management 43
CURRENT STATE OF THE PROBLEM 48
MATERIALS AND METHODS
Epidemiology 50 Clinical manifestations 53 Nutritional status 59 Role of infection 61 Pathology 90 Mechanisms of infection 102
RESULTS
Epidemiology 116 Clinical manifestations 121 Nutritional status 136 Role of infection 145 Pathology 172 Mechanisms of infection 191
DISCUSSION 208
CONCLUSION 218
APPENDIX l
Tropical ulcer questionnaire 220 Transport fluid 226 Medium for spirochaete isolation 227 Ingredients for PAGE 228 Cell growth medium 229
APPENDIX 2
Suppliers 230
BIBLIOGRAPHY 235 LIST OF FIGURES
Fig. 1. Distribution of tropical ulcer. Fig. 2. Schematic representation of ulcer progression. Fig. 3. Parts of a Hungate tube. Fig. 4. Cannulae for delivery of gas. Fig. 5. Method for preparation of transport fluid. Fig. 6. Hungate tube with anaerobic transport fluid. Fig. 7. Hungate tube showing loss of anaerobiasis. Fig. 8. Acute tropical ulcer. Fig. 9. Chronic ulcer. Fig. 10. Multiple tropical ulcers well separated. Fig. 11. Multiple adjacent tropical ulcers . Fig. 12. Squamous cell carcinoma . Fig. 13. Histology of squamous cell carcinoma. Fig. 14. Tropical ulcer scar. Fig. 15. Tropical ulcer and early leishmaniasis. Fig. 16. Histology of leishmaniasis. Fig. 17. Leishmaniasis in a child. Fig. 18. Fusobacterium type l colony. Fig. 19. Fusobacterium tupe l morphology. Fig. 20. Fusobacterium type 2 colony. Fig. 21. Fusobacterium type 2 morphology. Fig. 22. Electron microscopy of Fusobacterium type 1. (BHI ) Fig. 23. Electron microscopy of Fusobacterium type l. (FABA ) Fig. 24. Electron microscopy of Fusobacterium type 1. (WC ) Fig. 25. Electron microscopy of Fusobacterium type 2. ( BHI ) Fig. 26 PAGE of Fusobacterium types land 2. Fig. 27. PAGE of Fusobacterium types land 2, and other species of Fusobacterium. Fig. 28. Carbal fuchsin stain of cultured spirochaetes. Fig. 29. Histology of tropical ulcer showing spongiosis. Fig. 30. Histology showing vascular changes. Fig. 31. Dieterle stain showing bacteria. Fig. 32. Dieterle stain showing spirochaetes. Fig. 33. Electron microscopy of fusobacteria in the dermis Fig. 34. Electron microscopy of spirochaete in the dermis Fig. 35. Immunohistochemistry ( T-cells ) of infiltrate. Fig. 36. Immunohistochemistry (B-cells ) of infiltrate. Fig. 37. Vero cells in culture with medium. Fig. 38. Vero cells exposed to Fusobacterium supernatant. Fig. 39. Mouse model showing cutaneous necrosis. Fig. 40. Guinea pig model showing abscess formation. Fig. 41. Histology of experimental ulcer. LIST OF TABLES
1. Nutrition and tropical ulcer. 2. The role of infection in tropical ulcer. 3. Bacterial culture media. 4. Selective media for bacterial culture. 5. In-vitro cytotoxicity cell lines. 6. Bacterial strains for in-vitro cytotoxicity. 7. Bacterial strains for animal inoculations. 8. Mouse strains for animal inoculations. 9. Frequency of tropical ulcer in Papua New Guinea. 10. Number of tropical ulcers per location. 11. Number of ulcers per patient. 12. Anatomical sites of tropical ulcers. 13. Age distribution of patients with tropical ulcers. 14. Sex of patients with tropical ulcers. 15. Relationship of tropical ulcers and trauma. 16. Body mass index of patients with tropical ulcers. 17. Body mass index of ulcer patients and controls. 18. Dermatophyte infections in Fiji. 19. Number of swabs analysed per location. 20. Bacterial isolates from each location. 21. Number of isolates per swab. 22. Analysis of single isolates. 23. Frequency of multiple anaerobic isolates. 24. Bacterial species recovered from swabs. 25. Duration of ulcer and recovery of anaerobes. 26. Number of isolates from mud samples. 27. Biochemical profile of fusobacteria isolated. 28. Different characteristics of Fusobacterium species. 29. Characteristics of Bacteroides species isolated. 30. Pathology of tropical ulcer. 31. Patient serology - antibody levels to fusobacteria. 32. ELISA - antibody to bacteria from the ulcers. 33. ELISA - Rabbit IgG levels. 34. ELISA - Rabbit IgM levels. 35. Percentage of Vero cells affected. 36. Cell lines and cytotoxicity. 37. Cytotoxicity of supernatant fractions. 38. Butyrate concentrations causing cytotoxicity. 39. Inoculation of single bacteria. 40. Inoculation of multiple organisms. SUtf1ARY
Tropical ulcer is an acute or chronic skin disease seen in the tropics and subtropics that is characterized by necrosis of the epidermis and underlying subcutaneous tissue. The disease has been reported from China to the West Indies . Although it occurs mainly in children adults may also be affected. The disease often ha s a protracted course as the ulcers may continue for several months .
Previous reports on the disease, over the past 90 years, have included diet, trauma and infection as aetiological agents.
This study investigated one hundred and seventy-nine patients with tropical ulcers in five tropical areas. The nutritional status of the patients was assessed objectively . This study shows that there was no consistent relationship to overt malnutrition.
The clinical, bacteriological and histological features of the disease are also presented . The clinical findings were similar in all the areas studied. The acute ulcers ( less than six weeks duration ) were painful and oedematous whereas the chronic ulcers were relatively painless with a fibrous rim around the ulcers.
Histology was available on 20 patients and electron microscopy on 7.
The main findings were loss of epidermis, dermal oedema, a polymorphonuclear inflammatory cell infiltrate and blood vessel proliferation . There was no evidence of vasculitis in any of the sections . Bacteria were seen at the site of tissue damage.
l As previous reports have implicated anaerobic bacteria such as fusobacteria in the pathogenesis of tropical ulcers, a new method was developed for transporting samples anaerobically . Swabs were taken from patients in remote areas and transported to London for culture.
A number of bacterial species were isolated which included a new species of Fusobacterium. This new organism, F. ulcerans , could also be identified by ultrastructure of the histological sections.
This species of Fusobacterium is cytotoxic to Vero cells in-vitro and may account for some of the tissue destruction which occurs at the onset of the ulcers . This species of Fusobacterium itself does not produce an experimental ulcer, but when used together with coliform bacteria, also isolated from these tropical ulcers, an abscess resulted . This has been demonstrated in the guinea pig model which confirms that this is a synergistic rather than a simple polymicrobial infection.
2 INTRODUCTION
Study Plan
The aim of this study was to determine the cause of tropical ulcer.
The study was carried out by myself with assistance from
Dr . D. C. Robinson, a paediatrician, who was particularly involved in the epidemiology of tropical ulcers . The study took place in five tropical areas where tropical ulcer was thought to be endemic . A questionnaire was prepared and adhered to throughout the investigation to maintain uniformity . The questionnaire provided details of the patients personal, family and dietary history .
The survey was conducted over two years, and each area investigated for a month . Information about the living conditions and social attitudes of the patients in the areas studied, was obtained from local health visitors and nursing, medical or para- medical staff.
The clinical aspects of the disease were investigated and all the ulcers were photographed . The bacterial flora of the ulcers was studied and particular emphasis placed on the identification of the anaerobic flora . Fusiform bacilli have often been reported in smears, and have been implicated in the aetiology of tropical ulcers . However, these organisms have not been fully identified or characterised . As these organisms are anaerobes, detailed anaerobic bacteriology and the best method of sampling ulcers were undertaken.
Swabs were taken from the most recent ulcer for bacteriological culture . The bacterial samples had to be transported anaerobically
from the study areas to London . A transport system was devised to transport samples anaerobically and to ensure viability of the organisms.
3 As the ulcers develop rapidly, a toxic effect of the bacteria seemed a possible pathogenic mechanism for the infection. In-vitro cell culture systems were used to assess the aetiological role of the organisms isolated from the ulcers. A variety of cell lines were used to detect cytotoxicity. In-vivo synergy of bacteria to produce ulcers in guinea pigs was also studied.
The nutritional status of patients with tropical ulcer was assessed objectively to see whether the patients affected were malnourished.
Patients were also examined for other superficial skin infections, such as ecthyma, impetigo, scabies or fungal infections to see whether these conditions predisposed to, or were associated with the development of tropical ulcer. The role of person to person spread in communities was also investigated.
Skin biopsies were performed whenever possible. The sections were processed and stained with haematoxylin and eosin as well as special bacterial and fungal stains. The inflammatory cellular infiltrate was further characterised by means of a selection of monoclonal
antibodies and immunoperoxidase techniques.
The host response to the ulcers was assessed by an enzyme linked
immunosorbant assay, which used whole cell preparations of all the
organisms isolated frequently from the ulcers.
The hypothesis which this study hoped to fulfill, was that tropical
ulcer is a defined clinical entity due to a specific infection with
fusobacteria and spirochaetes.
4 Definition
Tropical ulcer has been defined in several ways . For instance it has been described as an acute sloughing ulcer which occurs on the leg below the knee . The lesion ma y be superimposed upon a wound or may arise apparently spontaneously (Clements 1936 ) . In 1932, Smith emph asised the description of "tropical sloughing phagedaena" as the foul necrotic slough left a raw granulating base which wa s surrounded by a raised, rolled sometimes indurated edge. Tropical ulcer has also been defined as a specific acute, ulcerative skin disease with a characteristic slough containing numerous fusiform bacilli and spirochaetes in the early stages (Blank,1947 ) . Hare
(1948 ) also described tropical ulcer as a speci fie disease associated with infection with the fusiform bacillus . Marsh (1945 ) quoted the definition used by Roy (1938 ) in the British
Encyclopaedia of Medical Practice which defined the disease as "a rapidly spreading ulcer, occurring on the lower extremities of the bod y, which quickly assumes a phagedenic character and is accompanied by considerable pain, local oedema, sloughing and a sero-sanguinous discharge." Kariks (1957) defined tropical ulcer as
"an acute, specific, inflammatory ulcerative process, involving the ski n and subcutaneous tissues , with characteristic adherent,
foul-smelling, slough overlying a soft, very tender and eas ily bleeding granulomatous base with surrounding oedema, caused by spir ochaetes and fusiform bacilli." This definition, like several of those ment i oned above, is broad and includes ·the clinical sites
aff ected as well as the aetiological agents .
5 All the definitions therefore include the common characteristics of these ulcers, namely that they develop rapidly and produce profuse amounts of foul-smelling slough which covers the friable base .
In this survey all patients who presented with an ulcer of rapid onset, which had a friable base covered with an exudate of slough, or those ulcers which had evolved through that phase and become chronic i.e. static in size and shape with a thick fibrous rim around the margins, were included . For the purpose of this study, tropical ulcer was defined as an acute or chronic skin disease characterized by dermal and epidermal necrosis associated with the rapid development of slough. This definition is based on the medical definition of "phagedenic" which refers to obstinate ulceration of the skin and subcutaneous tissues associated with sloughing and quick extension to the surrounding tissues (Butterworth) .
6 TROPICAL ULCER-REVIEW OF THE LITERATURE
The first cases of tropical ulcer were recorded in 1857 by Vinson in
Mozambique and those, together with all the other cases which were reported around that time were reviewed by Le Denteg (1899 ).
Tropical ulcer was described under several synonyms in the past.
These include Naga sore (Fox 1920 ) tropical septic ulcer
(Apostolides 1922 ) , ulcus tropicum (Panja 1945 ) and tropical phagedenic ulcer (Yesudian 1979 ) . Despite t he many synonyms, most writers believe that tropical ulcer is a definite entity . In the past the disease has been confused with several other ulcerative conditions which may occur on the leg and have a similar clinical appearance (Blank 1947 ) . These include streptococcal and diphtheritic ulcers and yaws . Burnie (1931) reviewed the other conditions i ncluded the literature as tropical ulcers . These include leishmaniasis, tuberculosis, syphilis and other "sores" . In his review he stated that several authors previously included different
t ypes of sores under the heading of tropical ulcer . Some patients with leishmaniasis for instance, were reported to present after
their lesions had developed into tropical ulcers . He thought that
most "sores" were caused through secondary contamination of minor
injuries, and were not due to specific infective organisms.
Similarly Adams (1923 ) regarded ulcus tropicum as a comprehensive
and uncertain term, to designate ulcers that terminate several
diseases such as syphilis, cutaneous tuberculosis, framboesia (yaws )
and leishmaniasis .
7 Epi demi ology of tropi cal ulcer
Geographical distribution
Tropical ulcer occurs widely throughout the world . The disease is found predominantly in warm countries and is particularly frequent in the tropics and subtropics ( Costa 1944, Loewenthal 1963, Ngu
1967, Golden and Padilla 1946 ) . Cases have been described from the
Caribbean (Clements 1936 , Earle 1942 ) and from Asia ( Yesudian 1979,
Fo x 1920 ) . Many reports from Africa confirm the existence of the disease on that continent. For instance cases were reported from
Uganda (McAdam 1966 ) , Nigeria ( Ngu 1967 ) , South Africa (Ferguson et al 1959 ) , the Gold Coast (Ampofo and Findlay 1950 ) and Kenya
(Jarvis 1945 ) . In Africa, the disease is seen most commonly in central Africa . Few cases have been reported from North Africa including Libya (Castellani 1948 ) .
The disease has also been reported from South America including
Bolivia, Brazil and Argentina (Costa 1944 ) , and much less commonly from Europe ( Clements 1936, Castellani 1957 ) . A map ( figure l ) outlines the distribution of the reported cases of tropical ulcer .
The hot, humid conditions were thought to predispose to the development of these ulcers, (Clements 1936, Kariks 1957 ) although it is known that tropical ulcers also occurred in the cold highland regions in Somalia (Adamson 1949 ) .
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9 Castellani (1957) however stated that tropical ulcer did not occur
in the temperate zone. Other reports on the distribution of the
disease, showed that the disease could occur in both dry and swampy
areas (Burnie 1931). These reports confirm that the disease may
occur in many areas irrespective of the climate or the degree of
humidity.
Prevalence
The prevalence of tropical ulcers is unknown because the figures
quoted by some authors refer to hospital admissions or patients who
attend out-patient clinics. In many areas where the disease occurs,
patients do not seek medical attention at all. Also in many
countries the hospitals are often situated in the major towns. Those
individuals with tropical ulcers who live in the rural areas never
attend these centres because of the great distance. A report of the
Sudan Medical Service for 1936 stated that 3% of all hospital
admissions for the whole country were patients with tropical ulcer
(Corkhill 1939 ) . The figure implied that the government figures
reflected the incidence of the disease in the country. However the
statement provides no information on the prevalence of the disease
in the country, only the percentage of those patients who were able
to attend hospital and were admitted for their tropical ulcers.
Several authors who have lived and worked in endemic areas recorded
their personal impressions. Such impressions are notoriously
inaccurate. Where figures are available tropical ulcer is a very
common condition in many countries. For instance, Burnie (1931 )
estimated that ninety-five percent of ulcers of the skin seen in
Kano, Nigeria were tropical ulcers .
•
10 Koerber (1950 ) in the former French West Africa reported an
incidence of 2. 6% in out-patients and 7% among in-patients
(Loewenthal 1963) . Thomson (1956 ) in northern Nigeria described
tropical ulcers as the commonest disease among the local
inhabitants. Nelson and Semambo (1956 ) examined 1,945 people family
by family in an area in Uganda where tropical ulcers were common .
Th ey found an incidence of 1% of the population affected with
ulcers . Similarly Ngu (1967 ) published figures from Nigeria and
reported an incidence of tropical ulcer in up to 33% of registered
hospital patients. Loewenthal (1963 ) reported that over half the
patients admitted to hospital in an area in Uganda between 1932 and
1933 had tropical ulcers. The same author also found that 2. 5% of
the Iteso tribe were affected with tropical ulcers and 13 . 6% of the
sam e tribe showed the typical scars of tropical ulcer . Until
population studies are carried out throughout the year on all the
persons in the areas where tropical ulcers are common only rough
estimates can be made of the prevalence of the disease.
There are well documented epidemics of tropical ulcer . For instance
Apostolides (1922 ) reported an outbreak of these ulcers in Palestine
in 1919. Up until that time ulcers were not seen in that area. In
As s am only isolated cases occurred up to 1942. Thereafter clusters
of cases developed and the dis ease became endemic in the area (Hare
1948 ) . The author attributed the influx of cases to a gradual build
up of infection among the population in the area as all other
conditions such as nutritional status, occupation and climate wer e
constant .
11 Seasonal variation of tropical ulcers
Reports on the seasonal variation of the disease are conflicting.
For instance Steinhauser in Aden, Smits in Sumatra and Chisholm in
the West Indies found that the prevalence of ulcers was greatest in
the wet, rainy seasons (Burnie 1931 ) . Similarly Corpus (1924)
reported that ulcers occurred more frequently in damp weather . On
the other hand Onarato (1927) attributed the high incidence in
Tripolitania to the drought and heat (Burnie 1931 ) whereas Burnie
(1931 ) himself reported an increase in the number of tropical ulcers
after the rainy season. Although Charters (1947 ) found an increase
in the incidence of tropical ulcers just after the rainy season he
attributed this to the lack of milk and fresh vegetables rather than
a seasonal variation in the prevalence of the disease. Similarly
Corkill (1939) and Dixon (1951) also noted an increase in the number
of cases just after the beginning of the rainy season but he also
ascribed this finding to the lack of vitamins available at the time.
Marsh and Wilson (1945) found that cold weather and rain favoured
the development of tropical ulcers . On the other hand Hare (1948 ) in
India found that tropical ulcers did not occur at all during the
cold, dry months. After the cold season no cases persisted. New
cases which de veloped thereafter were sporadic and thus unlikely to
be related to carry over of infection from the previous season. He
favoured an insect vector as an explanation for the seasonal
prevalence throughout the year .
Other authors have found that the prevalence of tropical ulcer is
constant throughout the year . Thus the report from Trinidad by Earle
(1942 ) , suggested that tropical ulcer occurred with almost equal
12 frequency throughout the year. He found the number of cases were only slightly increased during February to August when atmospheric temperature, rainfall and relative humidity were low.
The overall impression is that tropical ulcers can occur in any climate although the evidence presented seems to suggest that the disease is more frequent during the rainy season.
Age specific incidence
Tropical ulcer occurs more frequently in children than adults and those in their second decade have been reported to be most commonl y affected (Loewenthal 1963 ) . However other authors have suggested that the condition is most frequent during the third decade (Hughes
1931, Earle 1942 ) . Kariks (1957 ) stated that all ages can be affected and that age has little influence on the onset of the disease. In his experience the disease was seen most commonly in patients aged 15-45 years. Mo s t of those cases occurred in labourers who were usually adults. Hare (1948 ) reported that patients in the third decade were affected most often followed by those in their second and fourth decades. Most authors agree that the disease rarely affects children under the age of 5 years. This was possibly due to the fact that children were carried by their mothers and were
less exposed to trauma. However James (1938 ) stated that 2% of his
patients wer e under the age of 2 years. Most authors also agree that
tropical ulcers rarely appear for the first time in patients older
than 45 years although patients may develop recurrent ulcers over
for man y years.
13 Sex specific incidence
Earle (1942) reported that 67% of patients were male. He suggested that the difference reflected work practice. Men worked in oil
fields and were thus more liable to trauma. Similarly Mohanty (1945)
and Pattanayak (1944 ) described a male predominance of cases. Hare
(1948 ) also observed more male cases and stated that this was
related to the recruitment of males to the fields in the area where
he worked. Hughes (1931) went as far as saying that he had never
seen a case of tropical ulcer in a female. Kariks (1957), on the
other hand, felt that in the cities and on plantations sex may play
a role. Males rather than females worked in these areas and they
were more likely to sustain trauma which facilitated the development
of a tropical ulcer. However in the villages because women were more
actively involved in the agricultural work, and worked with men, the
incidence of ulcers was distributed equally between the sexes. In
contrast Loewenthal (1963) found a female predominance of cases.
Thus males and females ma y be affected and the number of cases is
most likely related to trauma sustained in employment.
Racial incidence
In the report by Earle (1942 ) negroes were affected more than any
other racial group. He attributed this increased incidence among
adult negroes to their employment in the oil fields where they were
constantly exposed to trauma. Children of negroid and Indian
extraction provided most of the juvenile cases. Individuals of
European, Chinese or American descent were relatively free of
ulcers. Hughe s (1931) in Malaya stated that the Chinese were
particularly prone to the disease.
14 Burnie (1931 ) found the disease almost confined to the natives of
tropical and subtropical countries and that only malnourished
Europeans developed tropical ulcers . Hare (1948) reported that he
had seen ulcers in civilian Europeans although this was rare . He
felt that in most cases their mode of dress or their work may have
predisposed them to attack by insects which he thought were vectors
of some infective agent . Although racial differences were observed
tribal differences were also recorded. Loewenthal (1963 ) in Uganda
noticed a difference in the incidence of the disease in people of
the same tribe. Those who were chiefs, policemen or house servants
wer e not affected even though they lived in close proximity t o the
res t of their tribe.
15 Clinical Mani festations of Tropi cal Ulcers
Tropical ulcers may occur as acute or chronic ulcers. The clinical manifestations which separate the two forms of ulceration are described .
The acute ulcer
Ulcers start with a tiny papule a few millimetres in diameter (Marsh
& Wilson 1945, Loewenthal 1963, Kariks 1957 ) . The initial lesion is
very painful . Although this stage is rarely seen by the attending
physician it may be observed when the patient develops a new lesion
while another ulcer is treated. The papule may become a vesicle
which contains sero-sanguinous fluid. Within a few days the
surrounding area becomes black due to tissue necrosis . The papular
lesion may become haemorrhagic or even bullous . Most commonly though
it becomes frankly pustular. A few days later the central area of
necrosis sloughs to leave an ulcer . Over the next few days the ulcer
rapidly enlarges in all directions, becomes very painful and a thick
purulent slough covers the base of the ulcer. At this stage the
ulcers are foul smelling and produce copious amounts of exudate
which attracts flies. The base of the ulcer is often friable and
bleeds easily on contact . The ulcer is usually round to oval, 2-6
centimetres in diameter and has a slightly raised edge and
surrounding hyperpigmentation. There may be some swelling around the
ulcer but the ulcers seem to provoke very little reaction in the
surrounding tissues .
16 Most of these acute ulcers remain unchanged without treatment and heal slowly but spontaneously over a period of up to eighteen months although on average they heal within in six months (Loewenthal 1963,
Earle 1942 ) .
The chronic ulcer
Except at the onset of the ulcers the lesions are rarely painful
(Nelson and Semambo 1956, Loewenthal 1963 ) . After the acute
ulceration and rapid enlargement the ulcers cease to extend. The
surrounding oedema settles and the edge becomes prominent. Often the
surrounding area forms a firm lip of fibrous tissue around the
ulcer . The base of the ulcer becomes less friable and the slough
diminishes ( Roberts and Highet 1986) . On average chronic ulcers
develop six weeks after the initial onset of ulceration . These
ulcers may take months or even years to heal. When healing finally
begins the edge flattens and granulation tissue gradually fills the
de fect . The subsequent scar takes up the shape of the original
ulcer. The scars are often atrophic and liable to break down .
Recurrent ulceration is not uncommon . The schematic representation
of ulcer progression is shown in fig . 2. At present there are no
ob vious features that predict which ulcers will become chronic .
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"f "f
.,. .,.
,t ,t
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/I( /I(
...... t~-'f"''l{ t~-'f"''l{
entatio
m
illustrating illustrating
papule papule
ecrosis ecrosis
yperpig
l l
scheme scheme
·~ ·~
, ,
n
h
~ ~
A A
= =
= =
4', 4',
......
. .
2
>,,, >,,,
Phase Phase
......
~·
Fig Fig
; ;
. .
......
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":]:...... ":]:......
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-
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'///////, '///////,
Preulcerative Preulcerative
h, h,
-;-~- .. ..
·:
~
• •
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-~.
CD CD I- Sites affected
Most ulcers occur on the lower leg, foot or toes. Less commonly the hand or arm is involved (Hare 1948 ) . Lasbrey (1952) observed
tropical ulcers on the nail bed of the toes . Any of the toes were
affected. Ulcers may be single or multiple. Where they are multiple
and exist side by side they have been referred to as "kissing
ulcers" (McAdam, 1966). Ulcers may be widely separated on the same
limb or may develop on the upper and lower limbs. The trunk is
rarely affected.
Systemic manifestations
Systemic signs are uncommon in patients with tropical ulcers.
Despite the extent of these ulcers there are few instances where
systemic features have been reported. When secondary infection
supervenes, regional lymphadenopathy occurs and there may be
considerable erythema and swelling at the site of the ulcer
(Apostolides 1922). Fever ma y be associated with the secondary
infection. In most cases though the disease remains localised to the
skin.
The frequency of extensive disease or systemic symptoms varies among
different populations. For instance O'Brien (1951) regularly found
bone and tendon involvement as a complication of these ulcers,
whereas Loewenthal (1963) rarely found these features in his
patients. O'Brien (1951) also observed fever and lymphadenopathy as
associated features in some cases.
19 Role of Trauma
Burnie (1931) was convinced that trauma played a large part in the aetiology of these ulcers. He stated that "there appears no doubt that the great majority of the ulcers originate at the site of superficial injuries such as slight skin abrasions produced by scratching, contused wounds, injuries caused by stones, thorns, guinea-worm infections and the like." He reviewed 250 patients with tropical ulcers and all except six ascribed the ulcer to trauma.
Over 32% of the new patients who sought treatment attended for tropical ulcers which had followed trauma. Most of these patients were adults who worked as railway construction workers.
Earle (1942 ) reported that any form of trauma which damaged the
epidermis could initiate a tropical ulcer. The injury could vary
from a crushing blow followed by a haematoma to a laceration. He
reported two cases of tropical ulcers which followed a dog bite. He
felt that scratches, abrasions, or punctures that followed
accidental injury by plants or nails could initiate an ulcer. A
nurse developed a tropical ulcer at the site where she accidentally
injured herself with a scalpel used to debride a wound in a patient
with a tropical ulcer (Apostolides 1922 ) . Earle (1942 ) also reported
that tropical ulcer could follow scabies, impetigo or mosquitoe
bites. Jarvis (1945 ) suggested that tropical ulcers could develop
after jigger sores especially those which affected the toes.
Many authors have stressed the correlation between trauma and
occupation. However, Hare (1948) observed that many of his patients
were heavy manual labourers but never experienced any form of trauma
at the site of the ulcer.
20 the Moreover when ulcers and abrasions developed on the same leg into abrasions remained clean and did not necessarily develop the tropical ulcers. He postulated that when an insect vector bit close soft granulation tissue of an abrasion, the infection remained vector to the surface and healed rapidly. Whereas when the insect and bit unbroken skin the infection remained deep. A vesicle formed of when it broke down a larger ulcer was visible. Thus some form For trauma (even minor ) was necessary for an ulcer to develop.
example an insect bite.
y of Kariks (1957 ) also thought trauma was important in the aetiolog some tropical ulcers. He reported that 94% of his patients recalled
form of trauma prior to the onset of the ulcer. In his patients O'Brien there was a close correlation between trauma and occupation. trauma (1951 ) emphasised how difficult it was to obtain a history of could from the patients. In his study, 181 out of 209 patients
recall a history of trauma.
in the The evidence presented suggests that trauma does play a role linked aetiology of tropical ulcers. Often trauma and occupation are
and the contribution of each is difficult to define.
21 Complications of tropical ulcer
1. Osteitis
Tropical ulcers may extend to bone although fortunately this is rare. Bone involvement was reported by Golden and Padilla (1946) and
Brown and Middlemiss (1956). Golden and Padilla reported marked radiological cortical opacity and thickening which encroached on the
medullary canal, periosteal thickening and fine radial lines which were interpreted as calcification. Exostoses which limit patients'
mobility, were reported by Nelson and Semambo (1956) . However, no X
rays were included in the report so periosteal proliferation cannot
be excluded. Despite many reports on the complications of tropical
ulcer osteitis is rarely reported which suggests that it is rare.
2. Tendon necrosis
Ulcers which extend deeply may involve and damage tendons. Once the
tendons slough, healing of the lesion is delayed and the result is
severe deformity and a painful scar (Jarvis 1945 ) .
3. Gangrene of the limb
Gangrene of a limb may follow tropical ulcers and has been obseved
by Carayon (1957 ) and Apostolides (1922) . Healing may subsequently
occur with extensive scarring but many patients will require
surgery. Other authors rarely reported this complication and it is
possible that tropical ulcers were confused by some investigators
with other conditions as the descriptions given were vague and did
not appear to fit the description of tropical ulcers.
22 4. Recurrent ulceration
Ulcers may recur at the same or a different site. The recurrent lesions may develop shortly after one lesion heals or a year or two
later. James (1938 ) suggested that recurrent ulcers in scars were
not uncommon because the previously damaged skin had a poor blood
supply and this predisposed to further ulcers. Another reason for a
recurrence was the continued presence of organisms dormant after the
lesion healed (Smith 1933 ) .
5. Squamous cell carcinoma
The malignant transformation of a tropical ulcer to a squamous cell
carcinoma is rare and may occur in up to 2% of ulcers (Vint 1935 )
The lesions become progressively heaped up, with vegetating growths.
Onl y one report describes the development of metastases to the groin
(Nelson and Semambo, 1956 ) . The authors observed 9 patients with
squamous cell carcinomas among 350 patients treated. The diagnoses
were confirmed histologically.
6. Tetanus
There is a theoretical risk of tetanus following contamination of
tropical ulcers although reports of this complication are rare ( Ngu
1967 ) .
23 7. Hepatitis B infection
Recently Tibbs (1987) reported an incidence of 26% for hepatitis B infection in patients with tropical ulcers in Kiribati (formerly the
Gilbert Islands ) . Of 47 patients with tropical ulcers tested for hepatitis B infection, 10 carried hepatitis B surface antigen and two hepatitis Be antigen. Hepatitis B surface antigen was detected in all the ulcer exudates from subjects with hepatitis B surface antigen in their blood. The same was observed with hepatitis B e antigen. These patients had open wounds and transmission from older
to younger siblings could have accounted for the high incidence of hepatitis B infection among siblings. Boys had a higher rate of
infectivity than girls. This was thought to be related to greater
contact during play between boys who were encouraged to play with
their peers outside the family whereas girls were often confined to
close contact with their mothers for several years .
24 Role of nutrit i onal status i n t ropical ulcer
Many authors have emphasised the role of malnutrition in the development of a tropical ulcer . Burnie (1931 ) reported that as far back as 1867, in the Abyssinian war, British soldiers who were well supplied with food remained free from ulcers whereas the Hindus who for religious reasons were not allowed to eat fish developed ulcers.
McCulloch (1928 ) described malnutrition as a major cause of tropical ulcers . He analysed the diet of those who developed tropical ulcers
and observed that their diet contained large amounts of glucose but
lacked protein and salt. He even stated that tropical ulcer was a
"dietetic ulcer" and felt that the role of infection was minimal.
Similarly Dalrymple (1928) felt that consumption of certain foods,
such as fresh meats, reduced the frequency of ulcers. In Kenya two
tribes were compared with regard to the frequency of tropical ulcers
and the inclusion of meat in the diet. According to Orr and Gilks
(1931 ) those who ate meat rarely developed ulcers. Loewenthal (1963 )
observed that animal protein and calcium were required to prevent
the development of a tropical ulcer .
Loewenthal (1932 ) also reported that the ulcers were related to the
availability of food. During episodes of drought usually associated
with famine, ulcers were much more frequent. He reported that two
tribes who lived under very similar socio- economic conditions had a
different incidence of tropical ulcers. The only difference in
behaviour which he could detect was that one tribe ate fish
regularly whereas the other omitted fish from their diets because of
tradition . He also observed that even though tribes lived in close
proximity to each other the incidence of tropical ulcers differed
among the different classes.
25 For instance chiefs, native troops and policemen rarely developed ulcers whereas the rest of the tribe often did . The implication was that those living under poorer socio-economic conditions developed ulcers.
Similarly Charters (1943 ) reported the prevalence of tropical ulcers among troops in East Africa. Of the East Africans admitted to the troops 0.2% (n=l ) had ulcers and that patient may have had necrotic bone on the shin rather than a tropical ulcer . However 49 % of
Somalis admitted to the troops had tropical ulcers . He thought that the only difference between these two groups of people was their
diet. In particular the East Africans ate more dried beans, ghee and
maize meal whereas Somalis refused to eat dried beans. He felt that
there was perhaps an essential substance in dried beans or ghee
which protected people from tropical ulcers . Later (1947) the same
author reported that milk prevented tropical ulcers. When milk was
in short supply the number of ulcers increased considerably whereas
when milk became available again they decreased . However none of
these studies objectively assessed a control population .
Other authors observed that malnutrition was not an essential factor
for the development of tropical ulcers. Marsh and Wilson (1945)
observed that all their patients with tropical ulcers were
malnourished, with dietary deficiencies similar to those reported by
other workers . However although they were unable to correct the
diets, mo s t ulcers healed albeit slowly. Similarly Buchanan and
Sanderson (1935 ) found that dietary therapy did not necessarily heal
ulcers . Patients failed to respond to a diet rich in vitamins.
Corkill (1939 ) treated his patients with oral vitamin
supplementation and used cod liver oil dressings on the ulcers .
26 The results showed that ulcers treated with cod liver oil dressings healed more rapidly. Oral vitamin A provided no further benefit and proteins, rather than vitamins, were thought to be more important for healing.
To quantitate nutritional deficiency Corkill (1939 ) used clinical signs such as the presence of black patches on the tongue and blueness of the gums as indicators of malnutrition. He scored all patients with ulcers and tried to correlate nutritional deficiency with both the number of ulcers and their recurrence rate. The
assumption was that those with recurrent or chronic ulcers would
exhibit greater degrees of malnutrition than those with acute or
recent ulcers. He found that patients with recurrent ulcers were
more likely to be malnourished and that ulcers healed more rapidly
in those less malnourished. However there were patients with ulcers
who had no signs of severe malnutrition. He concluded that although
malnutrition was an important factor it was not the only factor
which predisposed to the development of tropical ulcers.
Further evidence against malnutrition as a major factor in the
aetiology of tropical ulcers was recorded by Hare (1948 ) . He
examined several patients on the tea estates in India for evidence
of malnutrition and found no correlation between malnutrition and
the development of a tropical ulcer. He stated that "there is no
valid evidence that food deficiencies play any part in the aetiology
of tropical ulcer, and any apparent relationship is merely due to
the fact that the diets of the poorer classes in most countries is
imperfect and that it is the poorer classes who by their habits are
liable to contract infection". Finally, McAdam (1966 ) found that the
majority of patients in Uganda showed no evidence of malnutrition.
27 He was able to produce experimental ulcers by inoculating ulcer material in healthy subjects just as easily as in ulcer patients.
Role of Calcium and Vitamins
Several authors suggested that ulcers were related to a deficiency of calcium. Earle (1942) found that patients with tropical ulcers had low serum calcium levels. Loewenthal (1932 ) also found a low serum calcium in patients with tropical ulcers and reported an improvement after intravenous administration of calcium. In contrast
Pawan and Camps-Campins (1931 ) reported the results of calcium
levels in patients in Trinidad with a variety of conditions which
included tropical ulcers. They could identify two groups of ulcer
patients. The first appeared well except for the presence of an
ulcer. The second were clinically malnourished and their dietary
histories confirmed that they were chronically malnourished. Blood
calcium levels were always normal or even elevated in the first
group whereas they were always low in the second. Charters (1943)
experimented with dietary supplementation to see which nutritional
supplements would be most beneficial. Patients were given calcium at
a daily dose of 0.5 g intramuscularly or intravenously. Other
patients were given vitamin A in the form of cod liver oil. Some
patients were prescribed nicotinic acid (or coramine, the diethyl
am ide of nicotinic acid), others vitamin C or riboflavin in the form
of condensed milk. All the patients treated were compared with
controls. Sixty percent of controls improved. The author found that
vitamin C and nicotinic acid had no effect on the rate of healing.
Those patients treated with calcium and cod liver oil healed
rapidly. He observed that East Africans had a low calcium intake in
their diet yet they rarely developed tropical ulcers.
28 From these studies he concluded that the ulcers resulted from a deficiency of vitamin A. Yet despite the dietary shortage of this vitamin there were no other clinical signs of vitamin A deficiency such as xerophthalmia or phrynoderma. Similarly Scott (1941 ) reported a deficiency of vitamin A in Tanzania in areas where tropical ulcer was frequent. On the other hand Jarvis (1945) also in
Tanzania found that patients in that area who developed bony fractures healed very well whi ch suggested that they are not in fact calcium deficient. He suggested instead that the soil in the area may be calcium deficient.
Corkhill (1939 ) reported improved healing rates with topical cod liver oil. However this therapy made no difference to acute ulcers
although chronic ulcers improved. This did not therefore prove that
vitamin A was beneficial to all tropical ulcers. Blank (1947) found
that ulcers responded to therapy with vitamin B complex. By contrast
Hare (1948 ) found that the disease was patchy in distribution which
could not be related to dietary deficiencies but to variations in
the terrain and of the vegetation.
Role of zinc
The role of malnutrition in the development of tropical ulcer was
investigated by Watkinson et al (1985 ) . Patients with tropical
ulcers were compared with an age and sex matched control population
in the Gambia. Although most of the patients had low serum levels of
zinc many of the controls also had low values. Despite adequate
replacement therapy with restoration of the serum levels of zinc to
normal there was no improvement in the healing of these ulcers. The
study was well conducted and based on quantitative zinc levels.
29 The evidence that malnutrition predisposes to tropical ulcer is contradictory . Many of the studies were anecdotal and few assessed objective data . A summary of the reports on the role of malnutrition in the development of tropical ulcers is shown in table 1.
30
with with
observation observation
observation observation
observation observation
observation observation
observation observation
supplementation supplementation
supplementation supplementation
supplementation supplementation
A A
Quantitative Quantitative
Quantitative Quantitative
clinical clinical
clinical clinical
Zinc Zinc
clinical clinical
clinical clinical
clinical clinical
& &
& &
Calcium Calcium
Vitamin Vitamin
(TU) (TU)
ulcer ulcer
TU TU
effect effect
effect effect
effect effect
of of
no no
increased increased
no no
increased increased
no no
increased increased
increased increased
Incidence Incidence
tropical tropical
and and
A A
beans beans
, ,
Nutrition Nutrition
"salt" "salt"
Zinc Zinc
Fish Fish
none none
. .
1
dried dried
Calcium Calcium
Protein Protein
Dietarv Dietarv
Vitamin Vitamin
and and
Deficiency Deficiency
Ingredients Ingredients
in in
Table Table
1985 1985
1963 1963
1945 1945
1945 1945
1939 1939
1932 1932
1928 1928
Watkinson Watkinson
Loewenthal Loewenthal
Panja Panja
Charters Charters
Corkhill Corkhill
Loewenthal Loewenthal
McCulloch McCulloch
Author Author
l.,.J l.,.J f-' f-' Role of i nfecti on
s been The role of infection in the aetiology of tropical ulcer ha ) and emphasised by some authors (Earle 1942, Burnie 1931, Blank 1947 which discarded by others (McCullouch 1928 ) . The first report Dantec, describes the bacilli associated with these ulcers was by le on the Plaut and Vincent (1899 ) . He observed swarms of bacilli several smears taken from the surface of the sores . Sub s equently smears authors confirmed the presence of the fusiform bacilli in the and later (1909 ) the spirochaetes in the ulcers were recognised that (Clements 1936 ) . Many other authors subsequently suggested disease there ma y be more than one fusiform bacillus involved in the several process (Brienl 1915, Fox 1920, Hughes 1931 ) . Initially workers thought that the two morphological types were different later stages of the same organism (Burnie 1931 ) . The organisms were
identified as Spirochaeta schaudinni (spirochaete ) and Bacillus was fusiformis (fusiform bacillus ) . The presence of these organisms were thought to be indicative of acute ulceration. When organisms
scarce, the ulcers were usually of longer duration or the patients
had some form of treatment (Earle 1942 ) .
Webb (1946 ) noted clusters of ulcer cases as if in an epidemic
however there was no definite spread from one person to another.
Thus although cross infection from person to person was thought
likely this wa s not proven . Similarly Jarvis (1945 ) also reported
epidemic outbreaks .
which Kotrajaras (1982 ) reported an outbreak of endemic leg ulcers
resembled tropical ulcers . However they never isolated fusiform
bacilli from any of the ulcers .
32 Relation to oral sepsis
The source of the presumed infective agent was investigated by several authors. Ap ostolides (1922) and Clements (1936) both suggested that tropical ulcer might be related to infection from the mouth. Gordon Thomson (1956) suggested that eating with the hands allowed transfer of the organisms from the mouth to the legs. The organisms could then be scratched into the skin or introduced by insects. Clements (1936) investigated patients in New Guinea and found that 25% of tropical ulcer patients had chronic periodontitis.
Contrary to this Earle (1945) reported that whereas infection with
Vincent's organisms was common in northern Peru tropical ulcers were uncommon. Similarly Panja (1945) recorded that in his experience patients almost always had healthy gums. Apart from these clinical impressions Hare (1948 ) objectively studied seven tropical ulcer patients and seven controls who lived in intimate contact with the affected patients . He examined swabs from the ulcers, the skin around the ulcers and the gums of all the patients and controls. He found that swabs from all seven ulcers but from only one patient's skin surface showed fusiform bacilli . Only one ulcer patient and one control had fusiform bacilli in the oral swabs . No controls had fusiform bacilli in skin surface smears . He concluded that although
the mouth might be the source of infection in a small number of cases it was by no means the only or even the major source of
infection.
33 Fusi form bacilli
These bacteria were frequently reported from swabs of tropical ulcers (Clements 1936, Forbes-Brown 1935, Charters 1943). They have been regarded as normal commensals of the mouth and are found in normal healthy people (Finegold 1977). Marsh (1945) reported that the organisms were found on "dirty" skin of affected patients. The fusiform bacilli hav e also been considered to be of secondary importance in these ulcers, particularly as they were often associated with other organisms in smears (Ferguson et al 1959 ) . A report of an epidemic in South Africa documented the presence of streptococci, staphylococci and fusiform bacilli in ulcers. The authors suggested that the primary lesion was a pyoderma caused by streptococci or staphylococci. Lesions were secondarily infected with fusiform bacilli which caused extensive spread.
Hare (1948 ) performed smears on early lesions in the vesicular stage and noted fusiform bacilli. Yet they were rarely seen on histology.
He thought that the bacilli were not seen on the histology sections because they were present in small numbers. After 12-24 hours the number of fusiform bacilli increased considerably. In the 18 patients investigated either staphylococcal or streptococcal infections were common.
34 Spirochaetes
Several reports have documented the presence of Vincent's organisms in smears of ulcers (Clements 1936, Forbes-Brown 1935, Eggers 1915,
Jarvis 1945 ) . Eggers (1915 ) reported tropical ulcers from China. He observed six t ypes of spirochaetes in the ulcers he examined although the cases investigated included ulcers other than tropical ulcers. His descriptions include straight forms of spirochaetes which he regarded as variations of the spirochaetes. These may have been bacteria. Associated with these spirochaetes he described fusiform bacilli and bacilli with clubbed ends. Similar spirochaetes were also observed in patients with chancroid and gonococcal urethritis. Thus the relationship of the spirochaetal infection to tropical ulcer is unclear.
Combination of fusiform bacilli and spirochaetes
Most of the previous authors concentrated on a combination of micro-organisms as aetiological agents in tropical ulcer.
Apostolides (1922 ) produced transmission tropical ulcers using
"parcels of pseudomembrane" from cases of ulcero-membranous angina.
He postulated that the fusiform bacilli and spirochaetes seen on the pu s smears might be the causative agents. He was cautious when he
interpreted his findings and stated that other conditions such as
debility or malnutrition were prerequisites for the development of a
tropical ulcer. He wa s adamant though that the only causative agents
in this condition were fusiform bacilli and spirochaetes.
A list of the authors and the results of smears and cultures from
ulcers is set out in table 2.
35
None None
Aerobic Aerobic
None None
Aerobic Aerobic
Aerobic Aerobic
Anaerobic Anaerobic
Culture Culture
ulcers ulcers
ulcers ulcers
ulcers ulcers
ulcers ulcers
ulcers ulcers
cultures cultures
bacteria bacteria
from from
from from
from from
from from
from from
and and
of of
Smears Smears
Smears Smears Smears Smears
Smears Smears
Smears Smears
Source Source
Smears Smears
ulcers ulcers
tropical tropical
from from
bacilli bacilli
bacilli bacilli
spirochaetes spirochaetes
& &
fusiform fusiform
recovered recovered
fusiform fusiform
Gram-positive Gram-positive
& &
Pseudomonas Pseudomonas
bacilli bacilli
bacilli bacilli
implicated implicated
bacilli bacilli
m m
formis, formis,
and and
and and
Organisms Organisms
form form
. .
Fusi Fusi
sifor
2
cocci cocci
Spirochaetes, Spirochaetes,
Fu
Cocci Cocci Spirochaetes Spirochaetes
B. B.
Fusi Fusi
Spirochaetes Spirochaetes
Bacteria Bacteria
Table Table
1966 1966
1922 1922 1935 1935
1931 1931
1915 1915
1920 1920
Author Author
Burnie Burnie
Fox Fox
Eggers Eggers Apostolides Apostolides
Forbes-Brown Forbes-Brown
McAdam McAdam
°' °' \J.J \J.J Transmission experiments of tropical ulcers
Many workers have tried to produce an animal model of tropical ulcer . For instance Burnie (1931 ) reported that Lloyd Patterson
(1908 ) tried to inoculate material from ulcers intradermally, kept the inoculated sites bandaged and typical ulcers developed . Balliano
(1916 ) repeated the experiment but only fusiform bacilli were recovered in smears from the experimental ulcers . Subsequent experiments by Fox (1920 ) , Smith (1932 ) , Panja (1945 ) and McAdam
(1966 ) all showed an alleged aetiological role for the fusiform bacilli . However these organisms had not been characterised and the observations from experimentally produced ulcers were based on Gram stains of smears of pus. The aim of these experiments was to show that tropical ulcer was an infective condition .
Fox (1920 ) performed several experiments after his attempts to culture the organisms. He used an emulsion of the discharge from the ulcers and also mi xed cultures of cocci grown from the discharge of the ulcers . A pure culture of fusiform bacilli inoculated intramuscularly into a guinea pig failed to produce an abscess or an ulcer. Burnie (1931 ) tried to reproduce ulcers and used flies which were commonl y found near ulcers . He was unable to transmit the infection through thes e insects .
37 Fusiform bacilli: identification and cultivation
rods which are Fusobacteria are Gram-negative, obligately anaerobic may be almost caecal in pleomorphic in shape and size. Some bacteria Yet others may show a shape whereas others are long and filamentous. ends others appear central swelling. Although many have tapered laboratory culture but rounded. They are nutritionally demanding in with relative ease with adequate techniques they may be recovered not survive repeated from clinical specimens. Howev er many will are predominantly subculture when exposed to air. The fusobacteria but may be commensals found in human and animal intestinal tracts,
of the repiratory tract (Finegold 1977 ) .
spindle ) was probably The term Fusobacterium (Latin, fuso a thin spindle shaped first used by Knorr (1922) to define the long (1984 ) , the bacteria Gram-negative bacilli. According to Duerden plauti-vincentii originally described by Knorr as Fusobacterium bacillus ) should (as the organisms resembled those of Vincent's buccalis . The term probably be classified today as Leptotrichia spindle shaped Fusobacterium therefore now refers to Gram-negative
rods.
back to the early part Attempts to culture the fusiform bacilli date (1913) tried several of this century when Krumwiede and Pratt surface cultures. culture methods. These included shake and used agar contained Initially these were unsatisfactory. They then bottom of the dish between two halves of the petri dish with the with paper to placed in the inverted cover. Plates were covered covered with paraffin prevent air entering at the sides. The rim was partially successful. to prevent dessication. These methods proved
38 Contamination with aerobes still occurred but was limted to the surface areas. The addition of horse serum was essential for growth.
This was probably the first successful culture of the fusiform bacilli. Subsequently Fox (1920) had only limited success when he cultured fusiform bacilli from ulcers . He used the technique described by Krumwiede and Pratt (1913 ) .
As culture techniques have improved many more species of the genus
Fusobacterium have been recognised and characterized (Holdeman,
Moore and Cato 1977 ) . There are now 15 species recorded in the
Virginia Polytechnic Institute manual (1977) . The main characteristic common to all species of Fusobacterium is the production of butyric acid as the major end product of metabolism.
Although they may sometimes resemble the Bacteroides species the production of butyric acid differentiates them from the latter. Mo st species are sensitive to a wide range of antibiotics although there may be interspecies variation. In general most are sensitive to penicillin and neomycin and resistant to rifampicin.
The role of different species of Fusobacterium in disease has been reported by several authors (Moore-Gillon et al 1984, Ydenfeld et al
1982 ) . Of all the recognised species, F. necrophorum and F. nucleatum are most frequently reported in clinical infections. The
former causes necrobacillosis in man and animals, a disease associated with necrotic tonsillitis and post-anginal septicaemia
(Mitre and Rotheram 1982 ) . In man, oral and dental infections, as well as cerebral, hepatic and abdominal abscesses may follow infection with F. necrophorum (Finegold,1977 ) . Ovine foot rot is an example of a fusobacterial infection in sheep.
39 The sheep develop ulcerative lesions on the feet after being in wet conditions for a prolonged period. The disease is due to a synergistic infection with F. necrophorum, a diptheroid and
Bacteroides nodosus (Hine 1983) .
On the other hand F. nucleatum has often been isolated from the mouth where it is considered part of the normal flora (Willis 1977 ).
It is often recovered from this site and may play a role 1n periodontal disease . F. necrophorum produces haemolysin and leukocidin. Both may contribute to the pathogenicity of the organism. F. nucleatum is not haemolytic . Other species of
Fusobacterium are only rarely isolated from clinical infections.
Fusobacteria are difficult to recognise without special expertise as the morphology is not always spindle shaped as expected. Only F. nucleatum is regularly spindle shaped . This has to be taken into account when these bacteria are identified. Bennett and Duerden
(1985 ) have provided a good practical method for distinguishing the various Gram-negative anaerobic rods . These techniques were used in this study to facilitate identification of the fusobacteria.
40 Pathology of tropical ulcer
Harvey Blank (1947) reported that the characteristic histopathology is only seen in sections taken from an actively phagedenic part of the ulcer. The central slough which he described as a
"pseudomembrane" was a structureless mass of tissue which had undergone coagulation necrosis and the granulation tissue extended to the fascia. On the other hand Burnie (1931 ) examined 20 ulcers histologically and found no distinctive findings. Golden and Padilla
(1 946 ) reported the pathology of 24 ulcers seen in Guatemala. They described epidermal hyperplasia, pseudoepitheliomatous hyperplasia but none showed evidence of malignancy . Polymorphonuclear cells infiltrated the epidermis and ·in some instances formed microabscesses. Loss of pigment wa s thought to be more common in older lesions and late lesions showed no pigment at all.
The dermal changes described included oedema, loose connective tissue, dilated capillaries and haemorrhagic foci. Lymphocytes and polymorphonuclear leukocytes were scattered in the dermis. In the older lesions the dermis was devoid of inflammatory cells and showed considerable fibrosis. Nerves were not affected. The subcutis showed
a moderate number of inflammatory cells, mostly lymphocytes. There were foci of necrosis associated with foreign body giant cells. The authors (Golden and Padilla) also observed the arteries and veins were thickened although the latter less so. Larger arteries showed either marked muscular hyperplasia with narrowing of the lumen, or a mixture of hyperplasia and interstitial fibrosis. They felt that the vascular changes were primary and not a result of the ulcer as the changes were noticeable in early lesions, and also outside the areas of heavy inflammation.
41 They examined a control population who had post mortems for other reasons. No similar vascular changes were identified in the sites where tropical ulcers usually occurred. They also compared the changes in leishmaniasis, which was common in Guatemala, with those of tropical ulcers. No similar vascular abnormality was observed.They concluded that the vascular changes could be used to differentiate tropical ulcers from other ulcers which occurred in
Guatemala.
42 Anderson and Roberts (1932) also reported success with grafting and
emphasised the advantage that a large ulcer or a small ulcer took
about the same time to heal. When the graft took after sugery the
average hospital stay was 14.9 days. Of 154 cases grafted, 103
grafts were completely successful. In 25 out of the 154 cases, more
than half the graft took and only in 7 cases the graft failed.
In Uganda where tropical ulcers occurred commonly, Nelson and
Semambo (1956 ) described a form of surgery which could be performed
by an itinerant team of health workers. They excised the ulcer under
spinal anaesthesia then grafted the area with a split skin graft.
Patients received penicillin for a week before surgery and a week
after. They emphasised that extended trials of other topical
therapies were unnecessary and whenever possible, excision and
grafting should be carried out. Their results were impressive (72%
cure rate ) . Surgery was mainly indicated for patients with chronic
ulcers. Surgery cut down the number of visits to the health centres.
Patients who had been house bound for up to ten years with crippling
ulcers, which extended to bone and caused gross deformity, were
given a new lease of life after surgery. They also found that
patients could be grafted at the health clinics and did not have to
be admitted to the central hospital.
47 Management of tropi cal ulcer
Over t he years various treatments have been recommended for tropical
ulcers . Prevention of t hese ulcers was regarded as the most
important aspect of therapy. Charters (1943) recommended protection
of the feet and legs with boots and trousers. This suggestion has to
be taken in context with the prevalence of tropical ulcers in the
area studied. For instance a report by Capt . Fyvie, Medical officer
to the East African unit, stated that his troops wore only sandals
with no socks for six weeks yet none of the men developed an ulcer
despite several episodes of trauma (Charters 1947) . Therefore it
seems clear that trauma does not necessarily lead to tropical
ulcers . An additional factor such as infection is possibly
necessary.
Topical therapy
Before the development of penicillin a topical treatment with tar
was common (Anderson and Roberts 1932 ) . Topical therapy with dilute
solutions of hydrochloric acid, iodine or potassium permanganate was
often recommended (Apostolides, 1922 ) . The same author also used
topical antiseptics before removal of the slough from the ulcers
daily . Other forms of topical therapy included cod liver oil
dressings (Corkill 1939 ) , magnesium sulphate and glycerine paste or
copper sulphate (Earle 1942 ) . Jarvis ( 1945 ) reported the beneficial
effect of eusol for the initial stages of an acute ulcer which
contained a large amo unt of slough . As the lesions cleared he found
acrifl avine more s uitabl e .
43 Marsh and Wilson (1945) described the "lock-up II treatment for tropical ulcers. They used a variety of topical applications such as bismuth, whale oil, vaseline and zinc oxide. The ulcers were measured, washed or powdered and a layer of ointment applied to the ulcer. After a layer of vaseline was applied the leg was bandaged in a plaster cast. Ulcers healed in 7 weeks. They found that the rate of healing was proportional to the size of the ulcer, the site and, in their opinion, the dressing applied. A topical application of
BIPP (bismuth, iodoform and paraffin ) or ZIPP which contained zinc oxide instead of bismuth seemed the most effective for healing the
ulcers. However Earle (1942) reported no benefit from systemic bismuth unless syphilis coexisted. Jarvis (1945) confirmed the
plaster of Paris cast method of treatment in Kenya. He suggested
that if patients walked on the cast they could be restrained from
doing so by incorporating the bottom bar of the bed into the
plaster!
Lasbrey (1952 ) reported the beneficial effect of topical aureomycin
to tropical ulcers. The ointment was placed on the ulcer with a
piece of gauze. Dressings were changed every five days. He observed
a good response to this form of therapy which was cheap and easy to
apply. Local treatments used by populations where ulcers are
frequent include banana leaves, extract of the manchineel tree and
saliva (Earle 1942 ) .
Earle (1942) suggested that rest was invaluable for healing ulcers
and observed that absolute bed rest decreased the healing time for
tropical ulcers by half.
44 Systemic therapy
Intravenous neosalvarsan was recommended for severe ulcers
(Apostolides 1922) . Earle (1942 ) reported success with sulphonamides. His results were subsequently confirmed by Dostrovsky and Sagher (1943 ) . In 1946 Webb reported that penicillin was the treatment of choice for tropical ulcers. Up until then no systemic agent was uniformly successful for their treatment but ulcers healed
rapidly with penicillin.
Later similar reports on the response of ulcers to penicillin were
documented . Penicillin was used "experimentally" in a patient in
Iran in 1947 (Pinkerton ) . After 100,000 units of penicillin
administered intramuscularly daily for only three days there was
significant healing . Subsequently the author used penicillin on
further cases for up to five days with a beneficial effect . Not all
patients responded though . One of the patients had a temporary
recovery but the infection then spread up the leg and the patient
required surgery . Smears showed fusiform bacilli and spirochaetes so
the ulcers may well have been tropical ulcers which extended rather
more than is usual.
In 1950 Ampofo and Findlay reported the successful use of oral
aureomycin for tropical ulcers at a dose of 250 mg 4-6 times a day .
Treatment continued for 4 to 7 da ys . Within 72 hours there was
improvement and organisms were no longer visible on smears from
these ulcers . In some cases no organisms were visible after just 24
hours. They claimed that penicillin only worked if administered in a
large oral dose and found that aureomycin in a dose of 750 mg daily
for three days was as effective as penicillin in large doses .
45 Lindner and Adeniyi-Jones (1968 ) reported successful results with oral metronidazole. Yesudian et al (1979 ) also administered metronidazole orally. The patients received 400 mg three times a day . Within 48 hours patients reported relief from pain . All ulcers healed in two weeks. They concluded that metronidazole was very effective for tropical ulcers.
Su r gery
Surgery has often been advocated for tropical ulcers. Indications included:
- ulcers which failed to respond satisfactorily to topical therapy
- necrotic tissue which required surgical removal ( James 1932 )
- progressive disease where local surgery failed to halt the disease
process. Then amputation was performed (Apostolides 1922 ) .
Braithwaite (1930 ) treated the ulcers with curettage under general
anaesthetic. He curetted the area and cauterised it with pure
carbolic acid. Healthy granulation tissue formed in fourteen days.
If a large area was curetted he grafted the defect. Jarvis (1945 )
also operated on some patients with tropical ulcers and his
indications for surgery were similar to those described above . He
observed that if he scraped the ulcer base it was sufficient to
promote healing . Where bone or tendon necrosis developed, surgery
allowed more rapid healing of the ulcer.
46 Anderson and Roberts (1932 ) also reported success with grafting and emphasised the advantage that a large ulcer or a small ulcer took about the same time to heal. When the graft took after sugery the average hospital stay was 14.9 days. Of 154 cases grafted, 103
grafts were completely successful. In 25 out of the 154 cases, more
than half the graft took and only in 7 cases the graft failed.
In Uganda where tropical ulcers occurred commonl y , Nelson and
Semambo (1956) described a form of surgery which could be performed
by an itinerant team of health workers. They excised the ulcer under
spinal anaesthesia then grafted the area with a split skin graft.
Patients received penicillin for a week before surgery and a week
after. They emphasised that extended trials of other topical
therapies were unnecessary and whenever possible, excision and
grafting should be carried out. Their results were impressive ( 72%
cure rate ) . Surgery was mainly indicated for patients with chronic
ulcers. Surgery cut down the number of visits to the health centres.
Patients who had been house bound for up to ten years with crippling
ulcers, which extended to bone and caused gross deformity, were
given a new lease of life after surgery. They also found that
patients could be grafted at the health clinics and did not have to
be admitted to the central hospital.
47 CURRENT STATE OF THE PROBLEM
Tropical ulcer has been reported worldwide. Though much of the data is anecdotal it is clear that children and young adults are often affected and the sequelae may lead to long term incapacity due to contractures, bony exostoses and squamous cell carcinoma .
Tropical ulcer still affects many children in the tropics . The morbidity from these lesions is considerable as patients may have recurrent ulcers over many years. Many people are incapacitated by crippling ulceration of the legs and are unable to work . In man y instances the patients previously worked as subsistence farmers which involved heavy manual labour . The presence of leg ulcers limited their normal activities . Verbal reports from health workers in various tropical areas suggest the disease is still endemic in certain areas . This prompted the study into this disease .
In the Gambia for instance ulcers occur frequently among the rural population . Therapy is not easily availabl e to all because of the lack of treatment facilities in the rural areas . In some clinics run by voluntary organisations, penicillin may be available but, needles and syringes for administration are in short supply . Most of the reports on the role of malnutrition and infection in the aetiology of tropical ulcer are anecdotal, and few compared the results in the patient population with those of a control population in the same area . For this reason these factors were investigated to obtain objective data on the nutritional status of patients .
48 Although several reports allude to the fuso-spirochaetal nature of the disease the fusiform bacilli have not been identified or characterized. This is probably due to the lack of good anaerobic techniques which have only recently become available. These bacteria are fastidious anaerobes which require detailed anaerobic microbiology for their isolation. Also well-equipped laboratories are rarely found in the areas where tropical ulcers are common. For this reason samples for bacteriological investigation need to be transported over large distances. Anaerobic bacteria do not survive unless maintained under anaerobic conditions. The role of these
bacteria in the pathogenesis of the disease has not yet been assessed. Experimental infections were largely based on Gram-stain
appearances. An in-vivo model to demonstrate synergy between the
bacteria isolated from these ulcers was required to determine the
pathogenecity of the bacteria.
There are no data on the host response to the mixed infection with
fusiform bacilli and spirochaetes. The reason recurrent ulcers occur
has not been previously investigated. This study was therefore
performed to objectively assess as many of these parameters as
possible.
49 MATERIALS AND ME THODS
Epi demi ology
Patients with tropical ulcers were seen in five countries where tropical ulcers were said to be frequent namely Zambia, the Gambia,
southern India, Fiji and Papua New Guinea . (Patients were seen by
B. A. in Gambia and Fiji) . The study took place between April 1984
and April 1986. Consultations took place at hospitals, rural
clinics, health centres and villages . Details of the personal and
family history were obtained through the consultations as well as
details of the socio-economic conditions prevalent in the area. Dr.
Robinson and I designed a questionnaire to use throughout the study .
The questionnaire is included in Appendix la.
The following information was obtained:
age of patient
occupation or parent's occupation
duration of ulcer
history of previous ulcers
relation of ulcer to trauma
number of persons affected in the household
In the Gambia patients were seen at the out-patient clinics of two
large hospitals. These were at the Medical Research Council centre
in Fajara and the local government hospital situated in the capital
Banjul. In the rural areas patients were seen at the mission
hospital clinics at Sabayinda and Marakissa as well as the Medical
Research Council stations based at Farafenny and Kenaba . Village
visits were arranged at Sabayinda and Bassey which are inland
villages situated along the main river .
50 In Zambia patients were seen at the hospital in the Eastern Province at Katete and at rural clinics in a further six provinces served by
the Zambian Flying Doctor service. On a few occasions home visits were carried out in the villages .
In Papua New Guinea patients were seen at the mission hospital
clinics at Fatima and Sissano as well as the refugee camps at
Kamporotoro on the Western border of West Sepik.
In Fiji patients were sought in the hospital out-patient clinics in
Suva, Lotaka, Nadi, Savusavu, and at local schools and villages
throughout the main island and Viti Levu which is one of the smaller
islands. Many health worker clinics were visited in the rural areas.
In southern India (Madurai ) patients were seen at hospitals on both
sides of the city. Patients were also sought in the tea plantations
and at the Karagiri leprosy centre.
The age of the patient with a tropical ulcer was recorded . When
patients were uncertain of their age, every effort was made to try
and obtain the details of the age . For example by recollection of
past events or other circumstantial evidence of the age was sought
so that the data (particularly the anthropometric data ) could be
interpreted. Details of any previous ulcers were recorded . This
included the age at which a previous ulcer developed and the
duration of any previous ulcers.
51 i.e. two A household was defined as those who normally eat together. awa y at wives in separate huts count as two households. Children was described school were included. The main activity of the patient etc. in detail. e.g. desk worker, outdoor worker, farm labourer
treatment of Most patients attended the clinics specifically for because their ulcers although some were referred by health visitors in the of the research project. Most patients were English speaking information Gambia, Papua New Guinea and Fiji but whenever necessary both English was obtained via an interpreter who was well versed in
and the local language.
52 Cli ni cal manifestations of tropi cal ulcer
The number of ulcers present at the time of examination were documented . All ulcers were represented on the questionnaire by a drawing which indicated the site, size and presence of associated features such as cellulitis, gangrene or lymphadenopathy . The skin was also examined for evidence of previous ulceration which could be recognised by scars. Other concurrent superficial skin infections such as impetigo, ecthyma, scabies and ringworm were recorded on the questionnaire . All tropical ulcer patients had their teeth examined for dental caries. An age and sex matched control population was evaluated in a similar manner in each location. The controls were persons seen in the same clinics as the patients but who attended for other unrelated conditions, or they were inhabitants of t he same village. All ulcers investigated in the survey were photographed.
Where multiple ulcers occurred the most recent ulcer was included for bacteriological and histological assessment in the study. The older ulcers were documented and included in the diagram .
Differential diagnosis of tropical ulcer
Tropical ulcer may resemble ecthyma, leishmaniasis, sickle cell ulcers, Buruli ulcers, cutaneous diphtheritic ulcers and yaws, where the latter still occurs. Prior to the breakdown of the ulcer the swelling may resemble an area of folliculitis or furunculosis.
53 Ecthyma
Ecthyma is a pyogenic infection caused by streptococci or staphylococci. The infection usually follows an insect bite, an abrasion or a laceration. In the early stages of the infection the ulcer is superficial and ma y resemble the early lesion of a tropical ulcer. Ecthyma may occur at identical sites to those of tropical ulcer. i.e the leg, foot or thigh. The lesions differ clinically in
that crust formation is the hallmark of ecthyma whereas crusts
rarely occur in tropical ulcer. Ecthyma lesions are usually not as
deep nor as large as tropical ulcers. The former are usually 0.5 - 2
centimetres in diameter whereas the latter are often at least a few
centimetres in diameter. The natural history of the two diseases is
also different. Ecthyma usually settles in a few days or at the
outside a few weeks. Once the crusts are removed the lesions heal
well although scars are common. Tropical ulcers persist longer.
Usually several week s or even months. The histology of ecthyma shows
that the infection extends to the upper dermis and rarely affects
the reticular dermis. Gram stains show gram-positive cocci on the
surface of the lesion. Bacteriological culture of lesions almost
always yields group A streptococci or less frequently
Staphylococcus aureus .
54 Leishmaniasis
A range of lesions may occur in leishmaniasis . The surface of the lesions may crust, but often indurated, granulomatous lesions are present together with ulcers . In the early stages the clinical appearance of the papular lesion of leishmaniasis may appear almost
identical to the early lesion of a tropical ulcer . The early papular
lesions of both leishmaniasis and tropical ulcer may progress to
become pustular. Lesions of leishmaniasis tend to occur on the
trunk, arms, upper legs or face and are less frequent on the lower
legs . The subsequent course of the two diseases usually
distinguishes the two conditions . The granulomatous lesions of
leishmaniasis develop gradually whereas the papule of the early
tropical ulcer extends very rapidly over a few days to become an
ulcer.
The two lesions differ histologically . Leishmaniasis shows the
Leishman-Donovan bodies, with a chronic inflammatory infiltrate in
the dermis . The infiltrate consists of histiocytes and lymphocytes.
The overlying epidermis is acanthotic . The infiltrate may contain
histiocytic granulomas and giant cells. A Giemsa a stain readily
shows the organisms in early lesions whereas they are rarely
observed in older lesions . On the other hand tropical ulcer shows
true ulceration with a mixed dermal inflammatory infiltrate.
55 Sickle cell ulcers
Leg ulcers in sickle cell disease cause considerable morbidity to the affected population. The ulcers usually start after the age of
10 years with peak age of between 10 and 20 years. Sickle cell ulcers rarely occur for the first time after this age. The age range
for patients affected with tropical or sickle cell ulcers is very
similar. Clinically there are both similarities and differences
between the two types of ulcers. Sickle cell ulcers which may be of
two types (spontaneous or traumatic in origin ) almost always affect
the lower third of the leg around the malleoli. They are virtually
never seen above the middle third of the lower leg. Tropical ulcers
may be found on any part of the lower leg, the foot or the thigh, or
less commonly on the upper limbs. Both lesions are painful
initially. Epidermal involvement results in necrosis and ulceration
and follows dermal necrosis a few days after the onset of clinical
symptoms. On the other hand tropical ulcers start with a very
superficial papule and clearly involve the epidermis at the onset.
The two conditions differ in their geographical distribution.
Whereas sickle cell ulcers are the commonest form of leg ulcers in
children in the West Indies, tropical ulcers are rarely seen in that
area. In West Africa on the other hand tropical ulcers are more
common than sickle cell ulcers. The clinical course of the ulcers
differs in the two diseases. Sickle cell ulcers are more persistent
and respond poorly to systemic antibiotics whereas tropical ulcers
respond rapidly to the early administration of antibiotics. This is
probably due to the different pathogenic mechanisms involved in the
two ulcers. Sickle cell ulcers follow infarction of the skin due to
sludging of red blood cells in the vessels.
56 endothelial cell Histologically sickle cell ulcers shows proliferation in the small arterioles whereas tropical ulcers do not show any histological vascular abnormality. The bacteriology of sickle cell and tropical ulcers differ. MacFarlane (1986) reported that in sickle cell ulcers, cultures yielded predominantly aerobic bacteria although anaerobic cultures were also performed. They found
that S. aureus , Pseudomonas aeruginosa , and b-haemolytic
streptococci were most commonly isolated from swabs. In only 8 out
of 80 ulcers Corynebacterium diphtheriae was isolated and 4 of the
strains were toxigenic. Fusiform bacilli and spirochaetes are
usually seen on the smears of tropical ulcers.
Buruli ulcers
These ulcers are caused by Mycobacterium ulcerans and usually
occur in young children. The population of patients affected is
similar to those with tropical ulcer. The lesions differ clinically
and histologically from tropical ulcer. In Buruli ulcers, the
subcutaneous tissues are primarily affected. Epidermal and dermal
involvement follow secondary to this. Initially the lesions are
painful as in tropical ulcer but there is no papular or pustular
lesion to start with. Buruli ulcers extend rapidly, may become
circumferential and cause massive tissue necrosis as they extend.
The ulcers are large and may reach several centimetres in diameter.
Buruli ulcers are deeply undermined and one may probe many
centimetres under the lip of the ulcer. They are also irregular.
57 Although tropical ulcers also extend rapidly they are very rarely more than 3 or 4 centimetres in diameter. The ulceration extends from the epidermis to the dermis and rarely affects the subcutaneous tissues. The histology of Buruli ulcers shows extensive subcutaneous tissue necrosis with extension to the overlying dermis and epidermis. A Ziehl-Neelsen stain shows the causative organisms in those areas.
Other conditions which may be confused with tropical ulcer include; a ) varicose ulcer in the older patient
b) syphilitic gumma
c ) primary or tertiary yaws
58 Nutritional status of tropical ulcer patients
The nutritional status of the patients was assessed objectively to: a ) identify whether or not the patients with tropical ulcers were
malnourished and b) assess whether malnutrition contributed to the aetiology of
tropical ulcer.
The study did not aim to eradicate malnutrition, nor to put into effect measures to maintain good nutrition subsequently. To this end a detailed dietary history was obtained. Particular emphasis was placed on the consumption of first class proteins at the time of
onset of the ulcer. The availability of food, particularly proteins,
throughout the year and the type of food normally consumed was
documented. The overall clinical appearance of the patients was
recorded as well as any specific signs of overt malnutrition such as
follicular keratoses, glossitis and angular stomatitis.
Information about the socio-economic and cultural background of the
groups was recorded. Details were obtained on the food habits, the
cooking practises and the beliefs and taboos of the population in
the area studied . The information was obtained from the patients,
their parents or the local health workers. The information provided
an overall idea of the agricultural methods, predominance of cash
crops, food imports or exports and the purchasing power of the local
population. During the consultations information was obtained
regarding distribution and storage of food, especially during the
lean months.
59 The most appropriate techniques were mandatory for a survey on clinical nutrition so that flaws in the design or interpretation of the results were minimised. Standardization of survey methods have been emphasised and with this in mind the patients were evaluated using the criteria recommended (WHO tech. report 1977 ) .
All patients were examined and their weight (kg ) and height (metres) recorded. The nutritional status of the patients was assessed with 2 the formula Weight/ Height This is often referred to as the
"Quentelet's index" after an early proponent but is now referred to as the "body mass index". This correlates well with skin fold thickness (Thomas et al 1976 ) . The method provided a continuous quantitative scale for relative weight which facilitated comparisons of individuals with population norms. Measurements of nutrition which give an indication of acute or chronic malnutrition were applied to the same patients to see whether the results showed any
discrepancy. These included weight for height and height for age
(Rao and Singh 1970, Waterlow 1972 ) .
Treatment
Patients were questioned about the treatment of their ulcers,
particularly whether or not traditional medication was used and
whether saliva was placed on the ulcer. Patients were also asked at
what stage of the ulceration they sought medical attention, whether
medical therapy was sought for previous ulcers and how often they
could attend clinics for treatment.
60 Role of Infection
Bacteriology
Preparation of equipment prior to field trips
Before each field trip, 0. 25% Ringers solution was mi xed together . K. ) with 0. 25 % peptone water (Oxoid, Basingstoke, U were (peptone-Ringers) and dispensed into 5ml glass Bijoux. They then autoclaved at 121°c for 15 minutes . This was used to disperse
the exudate/ pus collected from the ulcers.
Cotton wool swabs were prepared using non absorbent cotton wool on
an orange stick . Groups of four swabs were wrapped in foil and then
autoclaved at 121°C for 15 min .
Transport medium to convey the bacteriological specimens was
prepared at the London School of Hygiene and Tropical Medicine.
Pre- reduced anaerobically sterilised (PRAS) peptone yeast extract
( PYG ) agar and broth were prepared by a method modified from the
Anaerobe Laboratory Manual (Holdeman, Moore and Cato 1977 ) and
dispensed into Hungate tubes (Macey 1972 ) . Hungate tubes were
supplied by Bellco Glass, Vinelands, New Jersey, U. S.A. The tubes
consisted of a glass tube (8" in length and 1/ 2" in diameter ) , a
butyl rubber stopper and a screw cap ( figure 3) . Once the tubes were
filled with medium, they were closed with the butyl rubber stopper,
and the system was then held in place by means of the screw cap. The
advantage of this system was that the medium could be pre-reduced to
provide the necessary anaerobic conditions .
61 After the tubes were closed, anaerobic conditions were maintained until the tubes were opened. The self-sealing rubber stopper allowed materials to be introduced or removed with a needle and syringe without causing disruption to the anaerobic conditions within the tube. The tubes also served as individual anaerobic culture systems which could be used in place of anaerobic jars.
Th e formula used for the transport fluid is listed in Appendix lb.
Sodium sulphoxalate (0.03% ) was added to the fluid to increase the anaerobic conditions.
Method for preparation of transport fluid
The salt solution was prepared and stored at 4°C until use. A
suitable flask ( just slightly larger than the volume of the
ingredients ) was chosen to allow only a small amount of space
between the top of the fluid and the top of the flask. An open
rubber stopper and a plastic bottle with a hole cut into the top to
allow steam to escape, served as a chimney. This ensured minimal
loss of fluid during the boiling stage.
62 Fig . 3. Parts of a Hungate tube
- 63 The peptone, rezazurin, trypticase, yeast extract, sodium sulphoxalate and distilled water were weighed or measured and boiled under oxygen free carbon dioxide (British Oxygen Special Gases
Division, U.K.). The gas was delivered through one of the cannulae from a multi-pronged delivery system (Don Whitley , Shipley, U. K. ) attached to the cylinder of oxygen free carbon dioxide. The other cannulae supplied gas to four Hungate tubes ( figure 4 ) . The medium was boiled until the rezazurin Eh indicator changed the colour of the fluid from pink to colourless. After reaching boiling point, the medium was cooled while oxgen free carbon dioxide continuously
bubbled through the medium. Cysteine was added to the cooled medium
to provide a further reduction of the Eh. Thereafter haemin and
vitamin K were added. The medium was allowed to cool further unde r a
constant stream of oxygen free gas.
While the medium was cooling the individual Hungate tubes were
flushed with oxygen free gas ( fig. 5) . When the medium cooled
sufficiently, 10 ml aliquots of the medium were transferred with a
glass pipette from the flask to each individual tube. (.Adequate
cooling of the medium prevented the formation of a vacuum. If the
medium was transferred to the tubes while still warm a vacuum could
have occurred and air drawn into the tubes when they were opened ) .
The tubes were then sealed with the butyl rubber stopper and screw
0 cap, autoclaved at 121 C for 15 minutes and were then ready for
use ( fig. 6 ) The agar tubes contained the same ingredients as , listed
above together with 2% Davis agar which was added during the boiling
stage.
64 Collection of specimens: Sampling procedure
Swabs were taken from all the ulcers investigated. Where multiple ulcers occurred, the most recent ulcer was included for bacteriological assessment. Sterile cotton wool swabs were used to take the swabs taken from the base of the ulcers, beneath the advancing edge. The early pre-ulcerative papules were deroofed and the swabs were taken from the base of the centre of the papule. The swabs were then placed in the sterile peptone - Ringers solution and vigorously stirred. An aliquot of the inoculated solution was aspirated with a 26 gauge needle and a 2 ml syringe. All the air in the syringe was expelled. The sample was then inoculated into a
Hungate tube with Peptone yeast glucose broth. A further 2 ml aliquot of the inoculated transport fluid was then aspirated and
inoculated into a Hungate tube with molten Peptone yeast glucose
agar at 45°C. All the inoculations were done through the
self-sealing butyl rubber stopper of the tubes after the top was
swabbed with 70% alcohol. The broth tubes were then stored at room
temperature due to lack of storage facilities. The agar tubes were
rapidly rotated to simulate the formation of roll tubes, as
described previously in the Anaerobe Laboratory Manual (1977 ) , and
stored at room temperature. The tubes contained rezazurin, as the Eh
indicator, which caused the straw coloured medium to change to a
pink colour as soon as anaerobic conditions were lost. This alerted
one to the loss of anaerobiasis (figure 7 ) .
Mud samples were collected from areas where patients with tropical
ulcer lived. The same technique was used to introduce the mud into
the Hungate tubes. Only broth tubes were inoculated with mud. The
samples were stored in the same way as the clinical specimens.
65 fi.9..:_ 4. Method for preparation of transport medium
66 Fig. 5. Cannulae delivering oxygen free gas to Hungate tube
67 Fig . 6 . Completes Hungate tube with anaerobic transport medium
68 ~ 7 . Hungate tube showing loss of anaerobiasis
69 Skin biopsies for culture
Skin biopsies were obtained from as many patients as possible.
Verbal consent for the biopsy procedure was obtained from the patients or their relatives if they were under the age of consent.
Biopsies were taken from the outer edge of the ulcer. After the area for biopsy was cleaned with an aqueous solution of Savlon ( I.C.I.,
Macclesfield, U.K. ) the area was infiltrated with 2% lignocaine with adrenalin (Astra, King's Langley, U.K. ) which was administered with a 26 gauge needle and 2ml syringe. The biopsy specimen incorporated a few millimetres of normal skin. A 5 mm disposable punch (Stiefel, High Wycombe, U.K. ) was used in most cases. A few elliptical biopsies were performed using a no. 15 blade and scalpel
(Gillete, Isleworth, U.K. ) . The wounds were closed with 3.0 silk
suture material (Ethicon, U.K. ) and the sutures removed a week
later. Half the biopsy material was homogenised with a glass
homogeniser ( Gallenkamp, Loughborough, U.K. ) and dispersed in the
peptone-Ringers solution. The inoculum was aspirated with a 2ml
syringe and 18 gauge needle. All the air was expelled from the
s yringe and the contents inoculated into a Hungate tube with Peptone
yeast broth as described previously. The samples were left at room
temperature.
Processing of specimens
Bacteriological specimens were transported to London from the field
and cultured under aerobic and anaerobic conditions. All the Hungate
tubes were opened and the plates inoculated in an anaerobic cabinet
(Don Whitley, Shipley, U.K. ) . Each sample of transport medium was
inoculated onto a set of plates as shown in table 3.
70 The plates were inoculated mechanically with transport medium by means of a spiral plater (spiral systems Inc 6740 Clough Pike,
Cincinnati). This method facilitated the recovery of individual colonies as a very small aliquot of medium was dispensed.
Two sterile plastic disposable cups were filled with 70% alcohol and sterile water respectively. These were used to clean the stylus of the spiral plater. An aliquot of transport medium was aspirated from each of the Hungate tubes and placed in a sterile plastic disposable cup. A volume of fifty microlitres of the inoculated transport broth was aspirated by vacuum. After the dispensing stylus was lowered onto the surface of the agar plates, the plates were mechanically inoculated. The stylus was cleaned with alcohol and water between
inoculations. After the plates were inoculated, transport fluid was streaked onto a non-selective plate with a plastic disposable loop
(Don Whitley, Shipley, U.K. ). A 5 ug metronidazole disc (May and
Baker, Dagenham, U.K.) was placed on each of the non-selective
plates. The plates for anaerobic incubation were incubated at 37°C
for up to five days in an anaerobic cabinet filled with 85%
nitrogen, 5% carbon dioxide and 10% hydrogen.
CO were transferred from Those plates which were incubated under 2 the cabinet to an incubator set at 37°c and supplied with 5-7%
CO . Plates were examined at 24 and 48 hours. 2
All those colonies which grew in an anaerobic environment, were
sensitive to metronidazole or which produced no comparable growth on
Columbia blood agar under aerobic conditions were regarded as
obligate anaerobes. These colonies were picked out and streaked for
single colonies onto a BHI non-selective plate.
71 Their colonial morphology and staining characteristics were examined after 48 hours and 5 days growth on the non-selective plate. All the anaerobic Gram-negative non-sporing rods were further tested with various antibiotics described below.
The Hungate tubes which contained agar transport medium were also opened in the anaerobic cabinet. The individual colonies which had grown in the agar by then were "picked out" with a sterile pasteur pipette and plated onto non-selective plates for aerobic and anaerobic incubation. The plates which were incubated anaerobically included a 5 ug metronidazole disc.
72 Table 3. Bacterial culture media
1) Brain Heart Infusion Agar (BHI) plates (Difeo, East Molesey,
U.K.) supplemented with cysteine, hemin, vitamin K (Holdeman,
Moore and Cato 1977) and 10% horse blood. This served as a
non-selective general purpose medium for comparison of growth on
antibiotic - containing media. This plate will be referred to as
supplemented BHI plate.
2) Supplemented BHI agar with 10% horse blood and crystal violet
1:1000 for the isolation of fusobacteria (Ninomiya, 1972 ) .
3) Supplemented BHI agar with 10% horse blood and rifampicin 7.5
ugr/ ml for the isolation of fusobacteria.
4 ) Supplemented BHI agar with 10% horse blood and kanamycin 1000
ugr/ ml and vancomycin 7.5 ugr/ ml for the isolation of
Bacteroides spp.
5) Supplemented BHI with 100 ugr/ ml nalidixic acid for
Gram-positive rods.
6) Columbia agar with 10% horse blood (Oxoid, Basingstoke, U.K. )
for the isolation of aerobic organisms.
7) Mac Conkey agar (Oxoid ) to differentiate aerobic Gram-positive
cocci and Gram-negative rods.
8) Sabouraud's medium (Oxoid ) for isolating yeasts.
73 Preparation of media
The following media were prepared for the isolation and identification of bacteria :
For aerobic incubation:
l ) Columbia blood agar with 10% horse blood
2 ) Mac Conkey agar
3 ) Sabourauds medium
Anaerobic incubation:
l ) Brain heart infusion agar supplemented with 10% horse blood
cysteine
vitamin K
hemin
2) Fastidious anaerobe agar supplemented with 10% horse blood
3 ) Supplemented BHI with vancomycin 7. 5 ug / ml and kanamycin
1000 ug / ml
4) Supplemented BHI with rifampicin 7. 5ug/ ml
5) Supplemented BHI with crystal violet 1 : 1000
6) Supplemented BHI with 2% oxgall (Oxoid ) .
74 For all the above media, BHI and Fastidious anaerobe agar (FABA )( Lab
M, Salford, U.K.) were prepared according to the manufacturers instructions. All antibiotics were filter sterilised. The medium was autoclaved and cooled and blood and antibiotics where indicated, were added. The crystal violet and oxgall were added prior to autoclaving the media. Media were poured into standard disposable plastic petri dishes (Sterilin, U.K. ) .
Several selective media were used to facilitate isolation of the various bacteria from the swab s . These are included in table 4.
75 Table 4 . Selective media for the isolation of organisms
Media Isolates
with S~o CO Air 2 1. Columbia blood agar (CBA) Staphylococci, Streptococci,
Proteus & Pseudomonas spp .
2. CBA with netilmicin All of above except Proteus
3. Sabouraud's medium Yeasts
Anaerobic incubation
1 . Supp . BHI All anaerobic cocci & bacilli
Facultative anaerobes
2. Supp . BHI + rifampicin Fusobacteria
3. Supp . BHI with Bacteroides
kanamycin and vancomycin
4 . Supp. BHI with crystal violet Fusobacteria
5. NOS media with rifampicin Spirochaetes
6. BHI with pectin and starch Spirochaetes
7. MlO medium Spirochaetes
8. Kelly's medium Spirochaetes
76 Spirochaete isolation
Several methods were used to isolate spirochaetes. The ingredients of the various media are included in Appendix le. The first method used was de scribed by Leschine et al in 1980. The medium was prepared as described. All the ingredients were used except the volatile free fatty acids, rabbit serum and sodium carbonate. The medium was prepared in bulk under oxygen free nitrogen, dispensed into individual pre-reduced Hungate tubes and then autoclaved. The last three ingredients were filter sterilized. Tubes were cooled and when the temperature reached about 45°C the last three ingredients were added. The ingredients were mixed, aspirated with a needle and syringe and the appropriate aliquots were dispensed carefully into each tube through the butyl rubber stoppers to prevent the introduction of air into the system. A filter sterilized solution of rifampicin was added to each tube to give a final concentration of
7.5 mg / ml. When required, the tubes were heated to just above 42°C which allowed the agar to melt. Transport fluid (0 .5 ml ) from each specimen was inoculated and the tubes rotated and incubated at
37 0 C for a week.
The second method used Brain heart infusion agar ( BHIA ) plates. The agar was prepared according to the manufacturers instructions and
10% horse blood was added. After the medium was boiled it was allowed to cool before the plates were poured. Once the BHIA set in
the petri dishes, filter papers were placed on the surface of the plate and aliquots of transport fluid were poured onto the filter
paper. The papers were sealed around the edges with autoclaved
vaseline to prevent organisms from entering on the sides.
77 The third method used MlD agar as described in the VPI manual
(1977). The medium was again prepared in bulk and dispensed into individual Hungate tubes after they were flushed with oxygen free nitrogen. Each individual tube was inoculated as described above.
Kelly's medium was prepared as descibed (Kelly 1971, Stoenner 1974 ) .
Individual Hungate tubes were prepared as described above.
Aliquots were taken from the transport fluid and examined under a dark ground microscope (Leitz, U.K. ) for motile spirochaetes.
78 Identification of bacteria
Staphylococci
S. aureus: This organism was recognised by the presence of
haemolysis around the colony and by its ability to produce
coagulase as described by Cowan and Steel (1974). Those
staphylococci which did not produce coagulase were regarded as
coagulase negative staphylococci. Further identification was not done.
Streptococci
All streptococci were identified by gram stain, as described by
Cowan and Steel (1974), and then tested with a STREPTEX kit
(Wellcome Diagnostics, Dagenham, Essex, UK ) . Their ability to grow
anaerobically was documented as well as the presence of haemolysis
around colonies. Anaerobic cocci were tested with the API 20A kit.
Coli forms
Gram-negative facultative anaerobes were identified using the
criteria described by Cowan and Steel (1974) . All isolates were
then tested with the API 20A kit for coliform bacteria (API Laboratories, Paris, France).
Those samples which contained Proteus species, were then grown in the presence of varying concentrations of gentamicin, tobramycin or netilmycin to inhibit the growth of Proteus. The minimal inhibitory concentration for each of these antibiotics was calculated using the method of Miles and Misra (Cruikshank, 1973).
- 79 - Detailed identification of the Proteus and Pseudomonas species was not carried out.
Biochemical tests
The following biochemical tests were performed on all Gram-negative rods:
l) Catalase production:
solution of H o was added to a colony taken from the 48 A 10% 2 2 hour culture of the organism. The presence of bubbles arising from the colonies indicated the presence of catalase production.
2) Oxidase production:
Blotting paper strips were soaked in a freshly prepared 1% solution of tetramethyl-p-phenylene-diamine hydrochloride in distilled water.
A colony of the organism to be tested was streaked onto the paper with a glass rod and the production of a purple colour was regarded as a positive result. Pseudomonas aeruginosa served as a positive control.
3) Carbohydrate fermentation:
The API 20A kit (API Laboratories, Monralieu, Vercieu, France ) was used for all the anaerobic gram-negative bacteria isolated. A heavy inoculum of the organism on a was collected on a cotton wool swab and dispersed in the API 20A medium supplied with the kit. Each of
the tubes was filled to the required depth using a sterile pasteur pipette. Mineral oil sealed the tubes to test for indole to prevent
evaporation of the indole. The completed strip was placed in the
plastic holder which contained some water to prevent dessication.
80 The strip was closed with the plastic cover and incubated anaerobically for 48 hours in the anaerobic cabinet. A BHI purity plate was streaked at the same time. Carbohydrate fermentation caused a colour change on the medium from purple to yellow.
The API 20E system (API Laboratories, Monralieu, France) was used for the Enterobacteriaceae. The instructions of the manufacturer were adhered to and the strip was incubated aerobically for 24 hours.
Anaerobic bacteria
Those bacteria which only grew under anaerobic conditions were regarded as obligate anaerobes. All the anaerobic Gram-negative rods were further tested for their: l ) antibiotic susceptibilities using the discs recommended by
Duerden et al (1976);
2) products of metabolism by gas liquid chromatography.
The antibiotics discs contained penicillin (2mu), kanamycin (1000 ug ) , vancomycin (7.5 ug) and phosphamycin (300 ug).
Chromatographic procedures for analysis of acid and alcohol
products of Gram-negative anaerobic rods
One of the characteristics of anaerobic bacteria is their ability to produce typical fatty acids which can be detected by gas liquid chromatography ( GLC ) . To detect these acids, cultures have to be acidified and extracted with ether. These ether extracts can be chromatographed. This technique was used to identify the
Bl Gram-negative anaerobic rods isolated from the ulcers. The methods used were described in the Anaerobe Laboratory Manual (p 134 ) . A
Pye-Unicam chromatograph, series 204 (Philips, Cambridge, U.K.) was fitted with a 1.5 x 4mm glass column and packed with Chromosorb W
(80-100 mesh ) . The chromatograph was equipped with a flame ioniser and was attached to a computer (PU 4180 computing intergrater,
Phillips, U,K. ) . The flow rates for hydrogen, nitrogen and air were
44ml / min, 40ml / min and 400 ml / min respectively. The temperature settings for the injection port, the detector and column were
250°, 250°, and 150°C respectively. The chromatograph was cleaned with methanol initially and in between samples to clear any residual gases. Known volatile and non-volatile acids were used as standards .
These were tested every day before any test samples. The retention
times of these standard fatty acids were recorded daily.
Preparation of samples for GLC
All the anaerobic organisms isolated were grown in Brain heart
infusion broth for 24-48 hours and the media analysed for the end
products of metabolism by gas liquid chromatography. The samples
were centrifuged at 3000 rpm for 10 minutes at 4°C. The
supernatant was treated in various ways before chromatography .
l ) with DUOLITE (BDH chemicals, Poole, U.K. ) and centrifuged again.
A fixed amount (3ul ) of the supernatant was injected into the
injection port.
2) acidified and extracted with ether. A fixed amount ( 3ul ) of
supernatant was injected into the injection port.
82 Calculation of fatty acids produced
The retention times of standard fatty acids were compared to those of the test samples. The amount of fatty acid produced was measured by the peak in the graph which recorded the amounts of volatile fatty acids produced. The area under the curve was quantitated by an intergrated computer and the results printed.
Conversion of threonine to propionate
A further characteristic of fusobacteria is the conversion of threonine to propionate. All isolates of fusobacteria were grown in broth culture with and without threonine. The ether extracts were of both cultures were chromatographed and the amounts of propionate compared as described in the Anaerobe Laboratory manual (1977 pl28. )
The Gram-negative anaerobic rods identified after these tests included fusobacteria and bacteroides.
Fusobacteria
All the fusobacteria isolates were investigated in detail to determine their species. The following additional tests were done on all fusobacteria:
l ) Aesculin hydrolysis
A freshly prepared 0.5% w/ v solution of aesculin and ferric citrate
0.05% w/ v were added to Fastidious Anaerobe Agar (FABA). Blackening of the agar around the colonies after 48 hours incubation indicated hydrolysis of aesculin.
83 2) Gas production
Stab cultures were performed into freshly prepared FABA with 1% glucose in glass tubes. Immediately after a heavy suspension of the organisms was inoculated into the molten agar, the caps were replaced and the tubes incubated aerobically for 48 hours. Gas production was shown by bubbles disrupting the agar.
3) Hydrogen Sulphide production
This test was included in the AP! 20A kit and was performed according to the manufacturers instructions. A precipitate near the
the cupule indicated H S production. base of 2
4 ) Extracellular DNAase production
DNA ase agar (Oxoid) with 3% yeast extract was heavily seeded with the test organism. Plates were incubated anaerobically for 48 h or until adequate growth was observed. The plates were flooded with a
10% solution of hydrochloric acid. A clear zone around the colonies indicated DNAase production. Staph. aureus NCTC 6571 was used with each test as a positive control.
5) Casein digestion
Casein methylene blue agar wa s prepared to the following formula:
Brain Heart Infusion Broth (Gibco, Uxbridge , U. K. ) 3.7%
Casein (BDH, Poole, U.K. ) 3.0%
Yeast extract (Oxoid ) 0.5%
Agar (Davis ) 2.0%
Aqueous methylene blue 1% w/ v.
84 The agar was seeded with a heavy inoculum of the test organism and incubated anaerobically for 48 hours. The plates were left on the bench until the blue colour returned and then observed for opacity around the colonies. Casein digestion occurred when the plate was flooded with a 10% solution of HCL and a clear zone appeared around the growth.
6) Nitrate reduction
A filter sterilised solution of potassium nitrate was added to FABA to give a final concentration of 1%. The solid agar was streaked with the fusobacteria isolates. Nitrate A (0.8% sulphanilic acid in
SN acetic acid ) and Nitrate B (O .S% a-naphthylamine in SN acetic acide ) solutions were added to 48 hour cultures. A pink colour indicated nitrite ions.
7) Nitrite reduction
A filter sterilised soution of sodium nitrite was added to FABA to give a final concentration of 0.001%. The presence of a pink colour as above indicated the presence of nitrite ions.
8) Indole production
This test was included on the API 20A kit. The cupule was sealed with mineral oil to prevent evaporation of the indole.
Guanine and cytosine ratio
Dr. H. Shah (London Hospital, U.K.) measured the G+C ratios of the fusobacteria by the methods described by Owen et al (1979 ) and
Marmur (1961 ) .
85 Soluble cellular protein electrophoresis
Different species of Fusobacterium may be identified by their biochemical reactions and also by their soluble cellular membrane proteins. All the isolates of the genus Fusobacterium which were recovered from the ulcers, were characterised by analysis of their cell membrane proteins with polyacrylamide gel electrophoresis. A strain of Streptococcus faecalis , isolated from a tropical ulcer served as an internal reference standard for all the experiments as it produced a thick band half wa y down the column. This allowed easy comparison of bands produced by other organisms. Bacteroides species isolated from the samples were examined in the same wa y.
Method
The method used was a modification of those described by Moore et al (1980 ) and Laemmli (1970 ) . The electrophoresis tank apparatus was supplied by Biorad (Watford, U.K. ) The cleaned glass plates were wiped with ethanol to remove any grease and then dried thoroughly.
While one of the plates lay flat on a table, a perspex spacer strip was placed at each edge. The spacer strips touched the edges at the top and bottom and abutted on the sides of the glass plate. A second plate was clamped to the first with a screw type clamp at each side which ensured that the glass plates reached as far as possible into the clamp. A rubber sealer in the grooves at the base of the plate holder provided a water tight seal.
86 The samples (fusobacteria, bacteroides and faecal streptococci ) used were prepared as follows:
l ) Isolates were cultured for 48 hours in Brain heart infusion broth supplemented with 0.1% calcium carbonate (BHI-C ) (for gram negative bacteria) and BHI-C plus 0.025% tween-80 for gram positive bacteria after a gram stain of the colony confirmed a pure growth.
2) Samples were transferred to a conical centrifuge tube and centrifuged at 8000 rpm for 10 minutes at room temperature. The supernatant was discarded.
3) 0.15 gr glass beads (0 .1mm diameter ) (Ballotini, Stanmore, U.K. ) together with 0.10 ml of 0.15 M Tris buffer at ph 7.0 + EDTA
(l.02gr/ L) were added to the samples.
4) Tubes were vortexed for 10 sec and placed on a shaker for 2 intervals of 2 minutes each.
5 ) Samples were heated in a water bath at 550C for 5 minutes to precipitate the cell wall structures which cause smudging of the protein patterns.
6 ) Tubes were then centrifuged at 8000 rpm for 10 minutes.
7) Powdered sucrose equal to 1/ 3 volume of the supernatant was added to each tube.
87 Preparation of gels
All gel solutions were prepared with autoclaved or degassed water.
The ingredients used to obtain an 8.5% gel are listed in Appendix ld.
With a pasteur pipette, the glass plates were filled with resolving gel up to a pre-marked line approximately 4 cm from the top of the plates. The acrylamide solution was layered with about 3 cm of water delivered from a Pasteur pipette and the gel was left to set for about l hour. When the gel had set, the layer of water was removed.
The stacking gel was added to the resolving gel with a pasteur pipette. The gel was layered up to about one inch from the top of the plates. A comb was washed with water and alcohol then dried and carefully inserted into the stacking gel to prevent entrapment of air bubbles under the comb. The stacking gel was covered with electrode buffer allowed to set (about 30min). After the stacking gel set the comb was removed and excess fluid removed. The wells were flushed with electrophoresis buffer to prevent wa vy bands in the gel. The samples were added to the wells and carefully covered with electrode buffer to the top of the glass plates.
The upper buffer reservoir was placed on top of the glass plate assembly with the rubber sealers fitted in the grooves. The locks were removed from the bottom of the plates attaching them to the plate holder and the locks were placed in the holes at the top to clamp the upper reservoir to the glass plate assembly.
88 The above apparatus was placed into the buffer tank fitted with a water cooling system and a magnetic stirrer . The tank was filled with approximately 3. 5-4 litres of electrode buffer.
Air bubbles underneath the glass plates were expelled with a modified pasteur pipette which forced buffer between the plates. The rema1n1ng electrode buffer was carefully poured into the upper reservoir tank . The safety lid was fitted and the apparatus connected to the power supply. The water cooler and magnetic stirrer were started and the apparatus set at 150 volts and 33 mA . current.
The gels were allowed to run for about 5 hours. When the dye front reached the bottom of the gel, the power pack was turned off and the glass plate unit removed and dismantled. The gel was carefully removed from between the glass plates. The gel was fixed in 12% trichloracetic acid for 30 minutes and stained in 0.08% Coomassie blue stain (Moore et al 1980) . The gel was destained with 10% glacial acetic acid in water (vol/ vol ) until a suitable colour was obtained . Gels were transferred to "ziploc" bags for photography and storage .
89 Pat hol ogy
Preparation of fixative prior to field trips
The fixatives for the skin biopsies included 10% formal saline and periodate-lysine paraformaldahyde (PLP ) (McLean and Nakane 1974 ) which were prepared and transported in aliquots in separate containers . Aliquots of 3% gluteraldehyde and l M cacodylate buffer were prepared and used as fixatives for skin biopsies obtained for electron microscopy .
Skin biopsy technique
Skin biopsies were obtained as described previously. After the specimens were divided for bacteriologic culture the remaining half was fixed in either 10% formal saline or PLP and submitted for histology . Eight biopsies were fixed in gluteraldehyde for electron microscopy .
Processing of biopsies
Light microscopy
Specimens for light microscopy were embedded in paraffin. Sections
( Sum ) were cut and stained with haematoxy lin and eosin ( H&E ) for routine histology. Special stains included the Giemsa, Gram,
Ziehl-Neelsen, and Periodic acid Schiff (PAS) for bacteria and fungi, and the Dieterle stain for spirochaetes, as described by
Pinkus and Mehregan (1981 ) . The sections were processed at St . John's
Hospital for Diseases of the Skin, London .
90 Immunohistochemical stains
Skin biopsies were dewaxed, and the inflammatory infiltrate assessed with the aid of T-and 8-cell markers (Dako, High Wycombe, U.K. ) using the method described by Taylor (1978 ) . Skin sections were also tested for fusobacteria as identified with an antifusobacterium antibody. Mangan and Lopatin (1983 ) reported that fusobacteria ma y stimulate 8-cell formation in-vitro. This was thus investigated.
Indirect Immunoperoxidase staining
An explanation of the method used is described below.
1) Tissue fixation may block immunoreactive sites. Enzyme pretreatment of sections "unmasked" the immunoreactive sites. This was carried out as follows:
Paraffin embedded slides were dewaxed and placed in running tap water for five minutes. Protease vll was diluted in PBS to a final concentration of 0.001% and placed on the slide for 15 minutes in a water bath at 37°C. The enzyme reaction was stopped by washing the slides under running tap water.
2) Endogenous tissue peroxidase activity was blocked by the addition of 1% hydrogen peroxide in methanol to the slide for 20 minutes. The slides were then washed in running water.
91 3) After the sections were wiped to remove excess PBS, 100 ul of the first antibody (antifusobacterium antiserum) was applied to the sections. The sections were incubated at room temperature for 45 min. The slides were then rinsed in PBS - tween and then in PBS.
4) The second antibody labelled with peroxidase (lOOul) was applied to the sections and the slides were incubated at room temperature for 30 minutes. The slides were again rinsed in PBS, followed by distilled water.
5) Sections were washed in water.
6) Solutions A and B were prepared for Peroxidase development with;
Solution A: 2.9 mls distilled water added to
0.1 ml Hydrogen peroxide
Solution B: 9 mls Tris buffer added to 1 ml stock DAB.
The developer contained 0.1 ml solution A which was added to solution B immediately before use.
The slides were flooded with developer and left for 5 minutes in a dark room. The reaction wa s stopped by placing the slides under running tap water.
7) Sections were counterstained with haematoxylin after washing with tap water.
8) A cover slip was fixed onto the slide with glycerol.
92 Electron microscopy
Samples for electron microscopy were fixed in 3% glutaraldehyde in
O.lM cacodylate buffer overnight. The following day, the biopsies were transferred into O.lM cacodylate buffer until they were processed in the department of Electron Microscopy at the London
School of Hygiene and Tropical Medicine. The samples were post fixed in 1% osmium tetroxide ph 7.2, dehydrated through graded alcohols and embedded in epoxy resin Epon 812. Semithin sections of 0.5 um were cut on an ultramicrotome and treated with lead acetate. The sections were examined with an AEl 801 transmission electron microscope at 60 and 80 kv acceleration voltages and 50 um aperture
(Wetherhed et al 1986 ) . For comparison, fusobacteria,
Enterobacter cloacae and Bacteroides spp. cultured in-vitro were examined in the same way.
Haematology and Serology
Venous blood samples
Whenever possible venous blood samples were obtained from patients with tropical ulcers and from a control population in the same area.
The blood was taken from the antecubital fossa with a 10 ml syringe and an 18 gauge needle. Haemoglobin estimation and electrophoresis were performed whenever possible. Samples were collected in an EDTA coated tube and gently rotated to prevent clotting.
93 A serum sample was collected from each venesection and kept in glass containers until the specimens were centrifuged (usually 4 to 6 hours later ) . Samples were centrifuged at 3000 rpm for five minutes.
The supernatant was aspirated with a pasteur pipette and stored at
4°C until departure. Serum samples were used to screen for anti cardiolipin antibodies with the disposable SYFACARD system (Wellcome diagnostics, Dagenham, U.K. ) . The sera were diluted to a titre of
1:8 and used according to the directions of the manufacturer. More specific tests to detect antibodies to Treponemes were not used.
Serum samples were also used to measure the antibody levels to the
organisms commonly isolated from tropical ulcers.
Patient serology
Most human sera contain antibodies to Fusobacterium as these
organisms are thought to be present in the alimentary tract (Hofstad
1974 ) . The specificity of these antibodies has not yet been clearly
established. Kristoffersen (1969 ) detected antibodies to a purified
protein antigen from an oral strain of Fusobacterium . Hofstad
(1974 ) examined human sera for antibodies which reacted with sheep
erythrocytes sensitised with lipopolysaccharide purified from F.
nucleatum. He found that all sera tested reacted with the
polysaccharide, although when he divided the sera into those
obtained from adults and children, the sera from the children
contained lower titres of antibody. The antibodies were of the IgM
class. This suggested that from birth, children exposed to the
continuous stimulation from antigens derived from indigenous
non-sporeforming, anaerobic bacteria produced antibodies to these
antigens.
94 The bacterial antigens could have reached the tissues through the intestinal mucosa or the non keratinized epithelium of the gingival crevices.
As patients were regularly exposed to these fusobacteria, antibody production in the case of an infection would probably of the IgG class ( Hofstad 1974 ) . Patients with tropical ulcers seem to develop recurrent ulcers for several years. This suggests that they do not mount a significant antibody response to overcome the infection. IgG antibody levels were measured by immunofluorescence in patients with tropical ulcers and controls. A whole cell preparation of the species of Fusobacterium isolated from tropical ulcers served as the antigen.
Immunofluorescence method to detect antibodies to fusobacteria
The antigen was prepared from a 48 hour brain heart infusion broth culture of Fusobacterium. The culture was centrifuged and the supernatant discarded. The deposit was washed three times with phosphate buffered saline (PBS ) and centrifuged at 10000 rpm. for 10 minutes at 4°C. A gram stain of the deposit showed there was >90% cell disruption. Glass slides were cleaned with alcohol and imprints made with a fixed prong inoculator dipped in glycerol. The glass slides were sprayed with teflon spray and allowed to dry. The
glycerol was washed off with hot water and the slides dried at
37 0 C for l hour. The whole cell preparation was diluted in PBS to
serial dilutions of 1:10, 1:20 and 1:40. Gram stains were performed
on all these dilutions to assess the concentration of organisms
present.
95 The most appropriate dilution was chosen for the experiments i.e. where the amount of cells was neither too small, nor too large. The slides were inoculated with the antigen using the same fixed prong inoculator. After the antigen dried on the slides in a dessicator
(Polysciences Ltd., Northampton, U.K.) the slides were fixed with acetone. The slides were prepared in bulk from the same preparation and stored at -70°C until required. This provided a standardised inoculum.
Four-fold dilutions of the patients ' sera were prepared from an
initial dilution of 1:16 (750 ul PBS and 50 ul serum ) . Six further
fourfold dilutions were made by adding 250 ul of the first dilution
to 750 ul PBS. This covered a range of dilutions from 1:16-1:16384.
The prepared antigen slides were fixed with acetone and allowed to
dry. Initially slides were also fixed with 1% hydrochloric acid to
assess the effect of different fixatives.
Aliquots of 25 ul serum from each dilution were added to each well
in duplicate. One well was left with only PBS as a control. Slides
were incubated for 30 min at room temperature then washed in PBS for
l hour on a shaker. The slides were then flooded with a
fluorescein-conjugate mi xture which contained:
2.6 ml PBS
0.3 ml Evans Blue
0.1 ml fluorescein labelled anti-human IgG conjugate (1:4 dilution)
(Dako ) .
The final concentration of the conjugate was 1:120.
96 Slides were kept at room temperature for 30 min. and then washed in
PBS on a shaker for 1-1.5 hours at room temperature. The slides were rinsed quickly in acetone and then PBS. The slides were mounted with glycerol and a cover slip. All slides were viewed with a fluorescence microscope (Nikon, U.K. ) and graded accordingly
(+to++++) .
Animal serology
Several biopsies from the patients with tropical ulcers showed
Gram-negative rods. The gross morphology was insufficient to separate the organisms especially since many cultures yielded facultative as well as anaerobic bacilli. An antibody to the
Fusobacterium was raised in a rabbit. With an immunoperoxidase technique this anti-fusobacterium antibody was used as the primary antibody to locate the fusobacteria and anti-rabbit IgG antibody was used as the secondary antibody.
Preparation of antigen and rabbit inoculation
The method used was described by Falkner and Hawley (1977 ) .
Forty-eight hour broth cultures of fusobacteria were used for the experiments. Each culture was centrifuged at 10000 rpm for 15 min
and washed three times with PBS. The deposit was suspended in PBS to
a dilution of 1:10. The amount of protein in the suspension was measured by the method described below. For each injection, 325 ug of protein was administered intramuscularly to the rabbit. The first
two injections, given a week apart were suspended in Freund ' s
complete adjuvant in a ratio of l part antigen to 2 parts
adjuvant.
97 The solution was mixed well and the injections given on the outer aspect of the thigh. A total of 6 injections were given the last 4 without Freund's adjuvant. The rabbit was bled from the ear two weeks after the last injection. An hour after the blood was collected it was centrifuged at 3000 rpm for 5 min and the serum kept at -70°C until required. Antibody concentration was quantitated by an enzyme - linked immunosorbant (ELISA ) technique.
Method for Protein Estimation
l ) The dye stock solution was prepared by mixing:
2 vol 85% H Po 3 4 l vol absolute alcohol
A 0.33% weight dye/ vol solution was prepared with 0.33 g Coomassie
dye added to 100 ml H Po and ethanol mixture. brilliant blue 3 4 The dye solution was kept in a dark bottle. The method used was
described by Bradford (1976 ) .
2) Standard protein (bovine serum albumin ) was prepared by
dissolving 0.5 gram bovine serum albumin in 50 ul bufffer solution
(PBS ) . The concentration of protein in the stock solution wa s
therefore 10 ug protein pe r l ul buffer. The stock solution was then
divided into aliquots and stored at -20° C until further use.
Dilutions of BSA
Protein ( BSA ) samples were prepared with 0.1 ml 1% BSA and 0.9 ml
water. Then 100 ul of diluted BSA were added to 400 ul water to give
a final concentration of 100 ug BSA / 500 ul water
98 Preparation of test solutions
The fusobacteria antibody was detected with
l) 50 ul test serum
2) a 1:10 dilution of the protein to be tested .
3) 50 ul control medium (SHI )
Preparation of dye reagent
The following ingredients were added :
Dye stock solution 3. 0 ml
Hl0 8 . 0 ml 85% 4 Abs . Alcohol 3. 8 ml
H 0 to 85.2 ml 2
Method
The mixture was filtered through a number one Whatman 's filter paper
into a brown bottle and covered with foil to prevent any light from
affecting the solution . The filtrate was brown in colour as compared
to the blue colour of the original mixture. The spectrophotometer
was standardised with buffer/ water (950 ul water with 50 ul buffer ) .
A representative sample of the dye wa s placed in a spectrophotometer
set at an optical density of 550 nm, to test the optical density of
the dye solution. The dye solution (950 ul ) was added to 50 ul
buffer in a plastic cuvette and allowed to stand for up to four
minutes before the optical density ( 0 . D. ) was read . An O.D. of
between 0.5 and 0.7 meant the solution was acceptable. If the value
was less than 0.5 a fresh solution was prepared .
99 The following concentrations of BSA were tested:
tube vol BSA ( ul ) vol buffer (ul )
l 0 50
2 5 45
3 10 40
4 20 30
5 40 10
6 50 0
Rabbit antibody production
The amount of antibody was measured by the enzyme linked immunosorbent assay (ELISA) . The following were prepared prior to the experiments:
l ) The test serum was diluted with PBS to cover a range of concentrations from 1:10 -1:160.
2) Substrate OPD was prepared by adding l ml ethanol to OPD. The mixture was vortexed well. Citric acid buffer ( 50 ml ) was then
added. The Citric acid buffer ( pH 9.6, 0.06M ) was prepared from;
0.38%
0 .192~~
3 ) PBS-Tween was prepared from;
Sterile distilled water 60 ml
6% Bovine serum albumin 10 ml
PBS-Tween x 10 6 ml
100 Method for Enzyme linked immunosorbent assay
A whole cell preparation of Fusobacterium antigen was used. The amount of protein in the antigen sample was calculated as described previously. The antigen was diluted with buffer (PBS ) to a final concentration of 2 ug protein/ 100 ul. Each well of a microtiter plate wa s coated with 100 ul antigen/ buffer and left overnight at room temperature. The following day the plates were washed three times with PBS-Tween.
Serum dilutions ( l:10-1:160 ) were added (100 ul ) to the wells. All tests were carried out in duplicate with normal rabbit serum as a negative control and a known positive sample as a positive control.
After 2 hours at room temperature the plates were washed three times with PBS-Tween. Antirabbit conjugate IgG (horse radish peroxidase)
(Dako ) was diluted in PBS to a final concentration of 1:10,000, and
100 ul added to each well. The plates were incubated at room
temperature for 2 hours.
The microtitre plates were thoroughly washed three times with
PBS-Tween and substrate-OPD added. The plates were incubated for 30
minutes in the dark. After incubation 10 ul hydrogen peroxide was 100 ul 2N H so . The added and the plates were fixed with 2 4 results were read with a Dynatek plate reader and integrated
computer.
101 Hechani sms of i nfecti on
Cytotoxicity
Tropical ulcers start as tiny papules and break down to ulcers very rapidly . This suggests that a toxic factor may be involved in the pathogenesis of the disease . This aspect of the pathogenes is was thus investigated with an in-vitro cell culture system.
Representative strains of four groups of isolates recovered from the ulcers were tested for cytotoxicity in-vitro . The test was based on the method described by Giugliano et al 1982 .
Table 5 shows the cell lines used which were obtained from the
Tissue Culture laboratory of the London School of Hygiene and
Tropical Medicine, London, U.K. The solutions required during the
test were prepared and are listed in Appendix le . These included two
types of growth support medium, medium 199 and Dulbecco's modified
Eagles Medium.
Preparation of test samples
Table 6 shows the strains used for the experiments . Two strains of
Fusobacterium isolated from the tropical ulcers, a strain of
Bacteroides fragilis , Enterobacter cloacae and Streptococcus
faecalis were included .
102 Table 5. Cell lines for in- vitro cytotoxicity
l) Vero (African green monkey kidney cells)
2) MRC 5 (human diploid lung cells)
3) Intestinal 407 (human embryonic intestine)
4) Chimpanzee liver
5) Chinese hamster ovary
Table 6. Bacterial strains for in- vitro cytotoxicity
l ) F. ulcerans NCTC 12111
2) F. ulcerans NCTC 12112
3) 8 . thetaiotomicron
4) E. cloacae
5) S. faecalis
103 Bacteriological media and culture conditions
from The bacterial strains used in the experiments were obtained for patients with tropical ulcers. The isolates were subcultured harvested purity and grown on BHI agar plates. The bacteria were glycerol from the purity plates, suspended in aliquots of peptone tested for and stored at -70°C until required. The bacteria and cultured cytotoxicity were recovered from the peptone glycerol or 48 h in 100 ml BHI broth at 37°C for 24 h aerobically always streak anaerobically ( as appropriate ) . A BHI purity plate was the broth inoculated simultaneously. Cells were harvested from . The culture by centrifugation at 3000 rpm at 4°C for 15 minutes (Millipore, supernatant was filtered through a membrane filter at -70°C 0 . 45u, Millipore, Middlesex, U. K. ) , and kept in aliquots was the until required. Uninoculated brain heart infusion broth medium the negative control for cytotoxicity and uninoculated growth
negative control for cell survival.
tested for The supernatants of all the fusobacteria isolates were
cytotoxicity after: 18-24 hours 1) The culture filtrates were dialysed against PBS for boiled in across a permeable dialysis membrane which was
distilled water for 15 minutes .
2) dialysed against PBS-with added cysteine (0 . 05 %) for 18-24 hours .
3) dialysed against polyethylene glycol for 18-24 hours.
4 ) heated at so 0 c for one hour .
5 ) heated at 80°C for one hour.
of 10 ml The treated supernatants were stored at -70°C in aliquots
until the experiments .
104 Method to demonstrate cytotoxicity
Cell monolayers were grown in either medium 199 or Dulbecco's modified Eagle's medium. After 24 hours when the cells had grown to confluence, the growth medium was decanted. Cell monolayers were stripped from the bottle with a mixture of trypsin and versene.
After the addition of trypsin and versene the bottle was gently rotated for 15 - 20 sec. The solution was decanted and the cells were incubated for 15 minutes to separate from the container. The cells were suspended in 10 ml medium and the cell concentration calculated with a haemocytometer. A sample of the medium which contained the suspended cells was added to the chamber with a capillary tube, and covered with a cover slip . All the cells were
counted in the 16 squares in each corner. The cell cou~t was
repeated from the diluted cell mixture . The cells were resuspended
in medium to give a final concentration of 300,000 cells/ ml.
Cytotoxicity tests were carried out in microwell plates (Flow
Laboratories, Rickmansworth, U.K . ) . Two-fold dilutions of culture
filtrates in test medium were prepared in 50 ul volumes to cover
dilutions from 1:2 to 1:256. These were transferred to the
appropriate wells. Then fifty ul of the cell suspensions were added
to each well using a multichannel pipette with disposable tips
( Titertek, Leighton Buzzard, U. K. ) . Plates were incubated for 24
hours at 37°C. All tests were carried out in duplicate.
Cytotoxicity was assessed morphologically at 18 and 24 hours . The
changes visualised were graded as+++ if 90% or more of cells were
affected;++ if 70-80 % cells were affected;+ if 50-60% cells were
affected;+/ - if 30-40% cells were affected, and - when < 20% cells
were affected.
105 Cells were fixed with buffered formaldehyde for one hour and stained with phosphate bu ffered crystal violet (0 . 0001% ) . Excess stain was eluted with 100 ul acid methanol ( 1% of l M HCL in methanol ) . The dye concentration was measured by reading the optical density at
540 nm on a Dynatec plate reader . The bound dye was used as an indicator of cell number (Barer et al 1986a, Barer et al 1986b ) .
All experiments were repeated on at least two separate occasions .
The five cell lines were tested individually with each of the test samples. Onl y Vero cells were used for further fusobacteria supernatant experiments .
Ga s Liquid Chromatography ( GLC )
Volatile fatty acids in the supernatants were analysed by GLC . The
technique and operating conditions were described previously . The
butyrate content in the Fusobacterium culture filtrates was
quantitated from the peak area under the curve . This value was
derived with an integrated computer ( PU 4180 computing integrater,
Philips, U.K . ) . Known concentrations of butyrate were used as
standards .
The role of protein and cytotoxicity
The role of protein in the supernatant, on the cytotoxicity to the
cell lines was investigated . The protein in the supernatant was
subjected to two forms of treatment . Firstly the protein was
precipitated with ammonium sulphate and secondly the supernatant was
passed through a column of Sephadex to see whether or not a specific
protein with a defined molecular weight caused the cytotoxicity .
106 A saturated solution of ammonium sulphate was added to an equal part of the fusobacteria supernatant to give a ratio of one to one. The mixture was shaken well and centrifuged at 10,000 x g for 10 minutes . After standing for one hour the supernatant was collected into a glass container and stored at 4°C until required . After the preliminary experiments showed that the cytotoxicity was lost after dialysis through a membrane which allowed passage of molecules of around 5000 MW, Sephadex G-25 beads (Pharmacia Chemicals, Hounslow,
U.K. ) were used in the column. The column, 100 cm long and with a diameter of 2. 6 cm, was fixed in a suitable place which avoided
draughts or direct sunlight .
The volume ( v) of Sephadex required was calculated from the formula 2 v= 11 r h.
One gram of Sephadex was equivalent to 5 ml volume. The Sephadex
beads were weighed and soaked in degassed PBS for three hours until
the beads were sufficiently swollen. The column was packed with
beads, and no air entered the column . The top of the column was
closed and the fine particles which had collected at the top were
decanted.
The column outlet was opened and the column was flushed with one bed
volume of PB S during which time the flow rate was monitored . A pump
adjusted the flow rate to between 10 and 20 ml / hour and the buffer
(PBS ) continuously flowed until the volume selected (2 bed volumes)
had been completed . The tubing was then connected to the
supernatant . A further bed volume of buffer passed through the
column after the supernatant.
107 The outlet of the column was connected to a fractionater, which mechanically collected and divided the eluent into a predetermined number of fractions, with a set volume per fraction .
The column was connected to a graph . The collection times of each fraction and changes in the molecular weights of substances eluted were recorded and expressed in an elution diagram . The variation of solute concentration in the eluent was detected by an ultra-violet monitor .
Experimental design for the repeat cytotoxicity studies
After the supernatant fluid was treated with ammonium sulphate and
gel filtration, the individual fractions were assessed for
cytotoxicity. For the ammonium sulphate experiments, ammonium
sulphate (one part ) and one part of PBS served as a control for
cytotoxicity and growth medium the control for cell survival . For
the gel filtration experiments, each fraction which showed an
increase in the solute concentration was used individually to test
for cytotoxicity. The original supernatant fluid served as a
positive control and growth medium as a negative control. All other
conditions remained the same .
108 In-vivo synergy
Several authors have attempted to assess the pathogenicity of bacteria isolated from tropical ulcers (Burnie 1931, McAdam 1966,
Smith 1933). Usually a mixture of organisms were inoculated but none of the studies were quantitative. In most cases pus from ulcers was inoculated into human subjects, or guinea pigs. The assumption had always been that organisms present in the pus could initiate an ulcer at a fresh site. These were therefore transmission experiments. Onl y a few experiments used pure cultures of organisms.
For instance, Fox (1920 ) used pus which showed only Gram-negative
rods and spirochaetes on the Gram stain. However a Gram stain did
not exclude small numbers of other organisms which were not seen on
the Gram stain, and which if unintentionally inoculated, could have
proliferated in the tissues.
Background to experimental work
The role of obligate anaerobes in serious infections has received
much attention in the last few years. Previously the . anaerobic
component of an infection wa s rarely thought to be important in its
pathogenesis. Anaerobes were often regarded as innocent bystanders.
However since anaerobic bacteria are frequently isolated from
clinical specimens, recent work has highlighted their importance in
clinical infections (Finegold 1977 ) . Anaerobic and aerobic bacteria
are often isolated together from specimens and frequently interact
to produce infections-yet each separately is not pathogenic. Various
animal models have been developed to evaluate this pathogenic
mechanism referred to as synergy.
109 1974, Kelly The intra-abdominal sepsis models (Onderdonk et al coli and B. 1978 ) clearly demonstrated synergy between ----E. Synergy has fragilis causing a abscesses in rats and guinea pigs. and post also been demonstrated in post operative wound infections studies of partum infections (Gorbach and Bartlett 1974 ) . Most numbers of bacterial synergy have involved the inoculation of small cavity of bacterial combinations into a wound or the peritoneal y between mice. Other reports also confirm the potential for synerg (Weinstein aerobic, anaerobic and facultative anaerobic bacteria a variety of 1975, Ingham et al 1981 ) . These experiments used pigs. Th e animal model s such as mice, hamsters, rats and guinea used, the success of the experiments depended on the organisms animal model and the method of inoculation.
abscess In this study animal experiments were performed to assess isolated formation by each organism or a combination of bacteria aimed to from the tropical ulcers (Hampp 1962 ) . The experiments animal model identify which bacteria could produce an abscess in an were isolated afte r subcutaneous inoculations. All the bacteria used commonly were from the tropical ulcers. Those bacteria isolated most
included in the experiments ( table 7) .
110 Table 7. Bacteria used in animal experiments
Streptococcus faecalis
Anaerobic cocci
Bacteroides thetaiotomicron
Fusobacterium ulcerans NCTC 12111
Enterobacter cloacae
Citrobacter freundii
Table 8. Mouse strains used in animal experiments
1. Balb/ c HEA2 (Dutch )
2. C57 black 10-BH2 haplotype
3. BlOR ( from C 57 )
4. CBA / Ca
5. STS / A
6. CXS Balb/ c x STS
111 Animals
All experiments were first performed with six to eight week old mice in the London School of Hygiene and Tropical Medicine animal unit.
The strains used are listed in table 8. The mice were housed and fed under conventional conditions. Two outbread strains of guinea pig, weighing 500-1000 g, were later used for the animal experiments. The studies were designed to develop an animal mode l simulating cutaneous ulceration.
Preparation of the inoculum
The bacteria were stored in peptone glycerol at -70°C. Strains were identified by Gram stain, biochemical tests and gas liquid chromatography (anaerobes ) . Bacterial suspensions were thawed to room temperature, subcultured for purity on BHI blood agar and incubated at 37°C for 48 hours in an anaerobic cabinet, or 18 hours at 37°C in 5% CO (aerobes ) . Broth cultures (100 ml BHI 2 broth ) were prepared for each of the test organisms, divided into
0.5 ml aliquots and stored at - 70°C until required. These were the stock cultures. Quantitative determinations of viable cell
density were obtained for all bacterial strains. Aliquots of each
species were used for 10 fold dilutions of aerobes and anaerobes in
BHI broth. Samples (0.1 ml ) of each dilution were plated onto
prereduced BHI agar plates, and incubated at 37°C for 24 hours
(aerobes ) or 48 hours (anaerobes ) . Colonies were counted and the
viable cell density was expressed as cfu/ ml. Bacterial stock
cultures were diluted to appropriate concentrations in BHI broth
(Cruikshank 1973 ) .
112 Experimental design
Mouse model
For the initial experiments the mice were shaved over the dorsal skin before subcutaneous injections were performed. All the isolates were inoculated singly into the skin of the back of the mice . The injections were repeated on at least two separate occasions and the reactions recorded .
Guinea pig model
The guinea pigs were shaved, the skin cleaned with 70% alcohol solution and the inocula injected into the back using a 18 gauge
needle . Inoculations were done into the dermis of the skin. In some experiments, a silk suture (Mersilk, U.K. ) was placed in the skin
prior to the inoculation. The behaviour of the guinea pigs was assessed daily. The wounds were inspected daily for induration,
erythema and abscess formation . A similar amount of brain heart
infusion broth without bacteria was inoculated as a control.
The guinea pigs were inoculated intradermally with a constant volume
of either a single pure organism or a combination of 2 or more
organisms . The size and type of lesion which resulted were recorded
and observed over one wee k, or less if the lesion was biopsied.
There were three groups of organisms isolated from the ulcers and
these were used in the experiments. They were coliform bacteria,
cocci and anaerobic gram-negative bacilli (A,B and C) . There were
thus seven combinations of organisms inoculated (A+B, A+C, B+C and
one of all three isolates A+B+C plus three of each organism alone ) .
113 Every experiment was repeated twice . The importance of each of the organisms was determined by its ability to cause an abscess or survive, or both when injected by itself, or by its ability to survive in a mixed infection which produced an abscess with other organisms.
Preparation of the sonicated fragment of fusobacteria
A 48-hour broth culture of fusobacteria was centrifuged at 10,000xg
for 10 minutes . The cells were washed three times with phosphate
buffered saline. The supernatant was collected and stored. The cells
were resuspended in PBS and sonicated in a sonicator ( MSE
homogeniser, MSE, Crawley, Sussex, U. K. ) with 8 bursts of 30 seconds
each, with a probe tip at 6 A . This produced more than 95% lysis of
the cells. The sonicated preparations were centrifuged at 2000 rpm x
g for 10 minutes and the supernatant fluid was discarded. The pellet
formed the sonicated whole cell preparation . The inoculum consisted
of 0.1 ml of the sonicated fragment (Falkner and Hawley 1977 ) .
Cultivation of abscess contents
Animal s were anaesthetised with intramuscular fentanyl (Janssen,
Wantage, U. K. ) once an abscess developed . The abscess was excised
and confirmed with haematoxylin and eosin stained histological
sections . The pu s from the abscess was diluted in one ml of BHI
broth and swabbed onto supplemented BHI blood agar with a 5 ug disc
of metronidazole and Columbia blood agar. Plates were incubated at
37°for 24 and 48 hours aerobically and anaerobically respectively .
Bacterial growth was documented and colonies of all organisms
isolated were identified by Gram stain and biochemical tests.
114 Histopatholoqy of experimental abscess
The skin biopsies were stretched to avoid deformation and fixed in
10% formal saline for at least 24 hours at room temperature. After fixation, sections were cut through the length of the skin biopsy and embedded in paraffin. Thereafter 5 um sections were cut and stained with haematoxylin and eosin and Gram's stain. These sections were processed at St. John's Hospital for Diseases of the Skin,
London.
115 RESULTS
Epi demiology
Areas surveyed
A total of 179 patients with 253 ulcers were interviewed and examined in five areas, namely Zambia, Gambia, Fiji, southern India and Papua New Guinea . The first survey was carried out in Zambia by
Dr . D. C. Robinson . Patients were seen at St . Francis Hospital,
Katete (Eastern province ) and in rural clinics in six other
provinces visited by the Zambian Flying Doctor Service. The second
survey was carried out in Gambia where patients were seen at the
major government hospital in Banjul and at the hospitals run by the
Medical Research Co uncil i n Fajara and Farafenny . Patients were seen
also in health care centres and rural clinics run by missionaries in
Sabayinda and Marakissa, and those run by the MRC in Bassey . In
southern India patients were seen at urban clinics at two hospitals
in Madras city and Ma du rai . I n addition patients were examined in
two rural areas :
1) The Schiefflin Leprosy Research and Training Centre at Karigiri
near Vellore, Tamil Nadu.
2) The Tata Tea Estate at Munnar, Kerala State .
In Papua New Guinea, four main areas were surveyed. These included
the Fatima mission ( West Sepik province ) , Sissano and Warapu in West
Sepik, t he Kamporotoro r e fu gee camp and the area around the towns of
Aitape and Wewak .
116 In Fiji patients were seen at the government hospitals in Nadi, Suva and Lautoka and health centres at Nausori and Vunindawa on the main island of Viti Levu . Patients were also sought on the smaller island
Vanua Levu at the coastal towns of Labasa and Savusavu. Village visits were made to the inland towns of Bua and Nabouwalu .
Social and environmental conditions
The patients examined in Gambia and Fiji mostly lived in rural villages of 10-100 houses . The huts were well separated and not more than 6 persons shared a hut at any one time . The size of the villages varied considerably with between 10 and 40 huts per village . On average each hut contained 2 rooms which were used as bedrooms. The huts consisted of thatch, leaves and dried mud. A few cement houses were scattered in a few villages but the majority of houses were of the thatched variety . In Zambia huts were similarly made of leaves and dried mud with corrugated iron as the roofing but many more were constructed out of mud based walls .
Water for drinking and washing was normally obtained from wells or
in some areas from taps located at varying intervals. The taps
(standpipes ) each served a population of between 20 and 100 people .
In Madurai the patients who developed tropical ulcer came from the
underprivileged areas where overcrowding was common . Water for
washing and drinking was supplied by street taps but washing was
also carried out in the river which could be reached only by
crossing wide bands of mud . The second hospital where patients were
sought in India, served the more prosperous part of the city . There
were no patients with tropical ulcers .
117 In the rural clinics of the Tata Tea Estate in the mountainous country around Munar in southern India, no cases of tropical ulcers were found even though the tea pickers walked barefoot and were exposed to frequent trauma . Tropical ulcer has rarely been seen at
Karigiri.
In Papua New Guinea, (Fatima, Sissano, Warapu) individuals lived in villages of up to 300 households with an average of five to six persons per household . By contrast the individuals in Kamporotoro refugee camp were living in very crowded conditions, often with several families in one house or tent.
In most areas, women washed inside the hut whereas men washed
outside . Overcrowding was seen in some areas but was not a constant
feature . Cooking was done outside in most instances . In most areas
individuals ate with their hands. Food was stored in the coolest
parts of the hut or occasionally in a paraffin-fuelled refrigerator.
Of the patients examined 95% were barefoot and all went barefoot for
most of the year. In most areas young girls wore long wrap round
skirts but also, for the most part, went barefoot . A small
percentage (12% ) said they sometimes wore shoes . Most (109/ 179, 61 %)
of the patients attended school . The presence of a tropical ulcer
did not interfere with their attendance at school and in many areas
dressings were don e at the village health centres after school .
Person to person transmission was not a frequent finding. All
patients were questioned about simultaneous involvement of other
family members. This was only seen in 9 patients in all .
118 Of 80 families s urveyed in t he refugee camp there were only two with more than one member who had an active ulcer . This suggests that person-to- person infectivity is uncommon .
In the Gambia most of the patients were muslim by religion so pigs were never seen in the villages . However cats, dogs, horses and chickens were commonly seen in and around the huts . Similarly, in the other areas surveyed, animals roamed freely around the huts .
Frequency of tropical ulcer
Currently there are no published figures available for the frequency
of the disease in affected areas . In Papua New Guinea the number of
patients seen during 1987 is listed in table 9. (Dr . G. Morris,
personal communication ) . The figures were compiled from the health
centres where treatments are carried out . In all other areas
surveyed there were no recorded figures available.
119
66 66
52 52
52 52
59 59
52 52 33 33
68 68
49 49
83 83 36 36
88 88
730 730
166 166
Total Total
------
6 6
6 6
9 9
0 0
0 0
25 25
23 23
14 14
11 11
18 18 29 29
12 12
Guinea Guinea
Ambunt Ambunt
New New
Papua Papua
in in
7 7
5 5
5 5
0 0
0 0
8 8
4 4
29 29
10 10 21 21
10 10
25 25
Maprik Maprik
ulcers ulcers
tropical tropical
3 3
30 30
36 36
30 30
28 28
34 34
34 34
18 18
14 14
19 19
45 45
17 17
of of
Wewak Wewak
Frequency Frequency
9. 9.
2 2
l l
2 2
6 6
7 7
9 9
8 8
4 4
32 32
30 30 14 14
30 30
Angoram Angoram
Table Table
Post Post
Aid Aid
Total Total
Jan Jan
July July
November November
September September
May May June June
March March
Feb Feb
August August
October October
April April
December December
District District
N N
D D ...... Clinical mani festati ons
The ulcers observed could be divided into acute or chronic ulcers on the basis of the duration of the ulcers . Those less than six weeks old were referred to as acute ulcers .
Acute ulcers
The pre-ulcerative lesions described by Hare (1948 ) were only seen in 6 cases. These lesions were small pustules no greater than 2- 3 rm,
in diameter . They were characterized by a raised, loose epithelium which was easily removed with a needle . This procedure was painless.
Below the epithelium an ulcer, less than a centimeter in diameter, could be seen . However the underlying ulcer had a greater diameter
than the overlying papule . These lesions were usually referred to as
"boils" or "pimples" by the patients. The pre-ulcerative lesion was
usually recognized by patients attending the clinic for treatment of
an existing ulcer . The pre-ulcerative lesions developed at any time
from 2 days to three weeks after the alleged episode of trauma . In
46 (26%) of patients the pre-ulcerative lesions occurred
spontaneously . The number of acute ulcers observed is shown in table
10 .
The ulcers less than six weeks old were all less than 5 cm in
diameter and showed the features of acute ulcers . They were round to
ovoid with a protuberant lip and a deep crater with a dense foul
smelling slough . The ulcers bled frequently so that dressings had to
be changed regularly . Almost all patients complained of pain at the
onset of the ulcers but this symptom only lasted from a few days to
about a week.
121 In 25% of cases the ulcer developed at a site of a previously recorded ulcer. In 65% of cases there was evidence of previous ulceration at a different site. The overall features of the acute ulcers were similar in patients from all locations (figure 8 ) .
Chronic ulcers
The chronic ulcers were less tender to touch . This was especially noticeable when the bacteriological swab was taken . These ulcers had
a thick rim of fibrous tissue round the outside of the lesion ( fig
9 ) . The ulcers ranged in size from 3-6 cm in diameter, except in one
patient where the ulcer was virtually circumferential (Robinson et
al 1987 ) . The base was less friable with less slough . The ulcers
lacked the foul smelling purulent exudate characteristic of acute
ulcers . The chronic ulcers gradually healed from the edges . The
ulceration extended only to the mid-dermis in the majority of cases.
Chronic ulcers were less common . Only 19 patients could confirm that
they had an ulcer for more than six months ( table 10 ) . Of these five
had had ulcers for more than a year . In two patients the lesions
started three years previously, healed after a year and then broke
down spontaneously over 6 months previously. However man y patients
(22, (12%)) were uncertain of the duration of the ulcer . Direct
questioning of the group uncertain of the duration of their ulcers
(as to whether the ulcer was present during the previous "wet
season" ) suggested that most ulcers were of several month's but less
than one years duration.
122 Number of ulcers
There were 121 patients with solitary ulcers but up to four were seen in a single individual . When multiple ulcers occurred they developed with equal frequency on the same or contralateral limb .
Multiple ulcers were for the most part well separated (fig. 10 ) although in a few patients (n=4 ) ulcers occurred adjacent to one another (kissing ulcers, fig.11 ) . Table 11 shows the number of ulcers for each patient .
Sites affected
The majority of ulcers occurred on the lower leg (81% ) . The sites of involvement are shown in table 12. Only the limbs were affected but the nail beds were never involved . In no patients was the trunk involved.
123 Fig . 8 . Acute tropical ulcer showing surrounding oedema
124 Fig. 9 . Chronic tropical ulcer showing thick fibrous rim
125 Fig. 10. Multiple ulcers on the hand and leg
(Courtesy of Dr. D. Robinson )
Fig. 11. Multiple adjacent ulcers on the leg
126 Table 10 . Duration of ulcers at time of examination
Duration Zambia Gambia India Fiji PNG
(weeks )
0-6 42 15 9 3 35
6-12 13 7 5 l 8
>24 3 8 l 0 7
Uncertain 6 11 0 0 5
Table 11 . Number of ulcers
Number Zambia Gambia India Fiji PNG
l 32 31 11 4 43
2 18 5 l 0 10
>2 14 5 3 0 2
127 Table 12 . Sites of Tropical ulcers
Zambia Gambia India Fiji PNG
No . ( %) No . ( ?~ ) No . ( ?0 No . ( %) No . ( ?0
Lower leg
Upper third 6 (7) 5 (7) 0 0 1 (1 )
Middle third 15 (17) 21 (32) 12 ( 55 ) 2 ( 50 ) 26 ( 34 )
Lower third 53 (62 ) 24 (36) 9 (41) 2 ( 50 ) 29 (38 )
Foot 11 (13 ) 13 (19 ) 0 0 15 ( 20 )
Upper leg 1 2 0 0 2 ( 3)
Upper limb 0 0 1 0 3 ( 4 )
86 65 22 4 76
Total number of ulcers : 253
Frequency at ulcer sites
Lower leg 81 ?~
Foot 15.4%
Upper limb : 1 . 6 ?~
Upper leg : 2 . O?~
- 128 - Age of the patients
Table 13 shows the age distribution of the patients. Most of the patients were aged between 5 and 15 years (96/179, 54%). In Zambia and India, more than 2/ 3 of the patients fell into that age range.
However in Gambia and Papua New Guinea a number of patients were older than 15 years (47). This difference can be accounted for by the predominance of adult women working as farmers in Gambia, or 1n
fishing in PNG. There were only 3 patients older than 45 years (2 in
Gambia, l in PNG ) . All three had several episodes of ulceration
previously and in all three the lesions had occurred in a site of previous ulceration.
Sex of patients
Table 14 shows the sex distribution of the patients. Males
outnumbered females only slightly except in India where there was a
significant male predominance. In Zambia and Gambia there was an
equal sex incidence. In Papua New Guinea females outnumbered males.
129 Table 13 . Age distribution of patients with tropical ulcer +------+ Age group Zambia Gambia India Fiji PNG (years ) +------+------+------+------+------+------+ I I M F Total I M F Total I M F Total I M F Total I M F Total I +------+------+------+------+------+------+
0- 4 0 0 0 l 3 4 0 0 0 0 0 0 0 l l
5-1 5 20 21 41 ( 64?0) 9 9 18 ( 44%) 9 l 10 (67?0) l 0 1(25?6) 12 14 26(47?6 ) ..... ~ D I > 15 13 10 23 ( 36?6) 10 9 19 ( 46%) I 5 0 5 ( 33?o) I 2 l 3 ( 75%) 111 17 28 ( 51?6) +------+------+------+------+------+------+ I I 64 I 41 I 15 I 4 I 55 I +------+ Total number of pat ie nts seen= 179. Table 14. Sex distribution of tropical ulcer patients
Se x of patients: Male ( ~~ ) Female 0~)
India 14 ( 93 ) l ( 7)
Papua New Guinea 23 ( 42 ) 32 (58 )
The Gambia 20 ( 49 ) 21 (51 )
Fi ji 3 ( 75 ) l (25)
Zambia 33 (51.5 ) 31 (48.5 )
93 (52 ) 86 ( 48 )
131 Complications of tropical ulcer
In only one patient (seen in Gambia ) an area of ulceration had undergone malignant change (fig . 12) to a squamous cell carcinoma.
This was confirmed histologically (fig 13). In four cases, however, there was clinical evidence of spread to bone . These patients were exquisitely tender to touch in the area of ulceration and adjacent tissues.
One third of all the patients investigated could remember a previous ulcer and 25% of patients thought that the present ulcer had
recurred in the same site. In a further 40% of cases, a history of a previous leg ulcer was confirmed by a large atrophic scar, which
took up the shape of the previous ulcer (fig .14 ) .
Relationship to trauma
Patients frequently reported a history of trauma albeit minor . A
definite history of trauma was obtained from 56 (31%) patients. The
types of trauma sustained are tabulated in table 15. The most
frequent injuries were associated with farming, walking in the bush
or playing outside . Other injuries resulted from falling off a
bicycle, playing soccer or patients were scratched by bushes . When
relatives accompanied the children, they confirmed the initial
injury. Only 5 patients thought that their ulcer was preceded by an
insect bite. The interval between trauma and the onset of the ulcer
varied from 2 days to three weeks .
~ 132 Fig. 12 . Squamous cell carcinoma developing
after a tropical ulcer
133 Fig. 13. Histology of Squamous cell carcinoma
Fig. 14. Scar of previous ulcer
- 134 - Table 15 . Injuries sustained prior to the onset of a tropical ulcer
Injury No . of patients
Playing soccer 9
Riding a bicycle 8
Wading in the river 6
Fall 18
Farming 6
Wal king outside 4
Insect bites 5
135 Nutritional status of tropical ulcer pati ents
Dietary habits of patients with tropical ulcer
Fish was freely available during the rainy seasons and in the Gambia and Fiji fish were regularly caught in the nearby rivers . Fish was frequently air-dried and the dried fish consumed during the dry and lean seasons . Fruit and vegetables were grown in most areas in Fiji but in Gambia only tomatoes and root vegetables were frequently obtained . Ground nuts was t he main produce of the Gambian farmers .
In PNG most of the patients were employed in fishing . Goat's milk was more readily obtainable than cow's milk but condensed mi l k was often used instead of fresh milk (Gambia) .
Assessment of nutritional status
The results for t he body mass index for 139 tropical ulcer patients are shown in the table 16. While the majority had values less than
19, that is below the qu oted desirable range for an American ad ult, no significant differences were found when tropical ulcer patients were compared to controls in each area as shown in table 17 .
Haemoglobin values recorded in Zambia and Gambia showed levels above
10 gr% in all cases . Only one patient in Gambia showed a sickle cell trait .
136 Table 16 . Body mass index of patients
No of ulcer pts
>19 normal (group A) 31
15-19 slightly malnourished (group B) 67
<19 grossly malnourished (group C) 41
139
137 )
)
27
32)
41
(
(
(
C
7
6 9
22
PNG 50)
(18) ( (32)
p
7
4
22
11
)
75
(25) (
3
C l
0
4
controls
)
------
Fiji -
(100
p
0
0 4
4
available
)
50)
50
(
with
5 (
5
C
10
)
0
India
(40)
(6
patients
p
0 0
6 4 10
of
status
35)
(46) (
(19)
C
5
9
26 12
)
23)
34)
42
( ( (
Gambia
Nutritional p
9
6
26
11
17.
)
)
37
56)
6
( (
(
Table
C l
6 9
16
)
25)
63
6)
( (
(
Zambia
p
2 4
10
16
A
B
C
------Group Group Group ------
vJ ...... CD No patients or controls were less than 75% of the predicted weight for height as calculated from the nomograms adapted from the Society of Actuaries (1959) and published in the report of the WHO Expert
Committee on Nutrition (1971 ) . Of those studied 80% were above the
90% value for the desired ratio . There was thus no evidence of recent or current malnutrition .
All those patients less than 18 years old were assessed for evidence of chronic malnutrition . The results showed no patients or controls had values less than 85% of the predicted values as calculated from the charts produced by Rao and Singh (1970 ) . There was thus no evidence of chronic malnutrition in the patients or in the controls.
Vitamin deficiencies
There were no clinical signs of vitamin deficiencies, particularly no angular stomatitis (riboflavin deficiency) follicular keratosis
(vitamin A deficiency) or follicular haemorrhages (vitamin C deficiency ) although subclinical deficiencies could not be ruled out.
None of the patients interviewed received concurrent treatment for either tuberculosis or leprosy. None of the patients showed overt signs of acute infections but chronic infections, such as malaria or parasitic disease, could not be excluded.
139 Association with other medical conditions
Although all patients were examined for other medical conditions or skin conditions there were no patients with evidence of either cutaneous tuberculosis or leprosy. One patient had clinical and histologic evidence of concurrent leishmaniasis and a tropical ulcer. Other skin diseases such as dermatophyte infections, impetigo and leishmaniasis were common.
Impetigo wa s present in 28 patients (16% ) . No patients received specific treatment for this condition. In Fiji, tinea imbricata was endemic. Large sections of the population were affected and the disease affected several members of the family. The diagnosis was
confirmed by culture in 37 cases. None of the patients with positive
fungal cultures had tropical ulcers. Tinea capitis and corporis were common in Fiji and Gambia. Children were affected more often than adults. In Fiji the causative organisms were T. rubrum or
T.tonsurans ( table 18 ) .
Leishmaniasis was seen only in Gambia. The early lesions were
identical to those of early tropical ulcers. One patient had a
tropical ulcer on the lower leg and an early papular lesion adjacent
to the ulcer showed histological features of leishmaniasis (see figs
15 & 16 ) . The patients with Leishmaniasis were older (average age 26
years ) although there wa s one patient only 4 years of age who showed
the characteristic clinical features of leishmaniasis (fig 17 ) .
140 fi.g_. 15. Tropical ulcer adjacent to leishmaniasis
141 Fig. 16. Histology of Leishmaniasis papule
•
- 142 - Fig . 17 . Leishmaniasis in a child
143 Table 18. Dermatophyte infections in Fiji
Source of sample Fungus cultured
Scalp hairs (1 ) T. tonsurans
Trunk (17) T. rubrum (6)
T. tonsurans (3)
Palm (1 ) T. rubrum
144 Role of infection
Bacteriology
Table 19 shows the number of swabs analysed from each location.
The results of the bacterial cultures from the swabs of the patients are summarized in table 20 . From the aerobic cultures, E. cloacae , coagulase negative staphylococci and faecal streptococci were isolated most frequently . Of t he anaerobic bacteria, fusobacteria and anaerobic cocci were isolated most often .
There were a total of 376 isol ates (240 aerobes and 136 anaerobes ) , which represented an average of 2. 52 isolates per specimen (1 . 6 aerobes and 0.9 anaerobes ) . Several swabs had more than one organism present . The detailed analysis of the number of isolates is shown in table 21 .
Where only one isolate was obtained, the organism present varied as shown in table 22 .
145 Table 19 . Number of swabs analysed from each location
Country No. of Patients No. of Controls
swabbed swabbed
Zambia 34 0
Gambia 41 3
India 15 2
Fiji 4 0
PNG 55 0
Total 149 5
146 Table 20 . Bacteriology of cultures ( from ulcers )
Aerobic culture
Gram-positive
Organism Gambia Zambia India Fiji PNG Total
Staph . aureus 1 1 2 2 6
Coagulase negative 17 9 8 3 14 51
staphs ( C1 S)
Streptococci 23 8 2 3 11 47
group A 0 0 0 0 0
D 23 8 2 3 11
104
147 Table 20 continued
Gram-negative
Organism Gambia Zambia India Fiji PNG Total
Enterobacter 25 12 3 l 13 54
cloacae
Eschericia 2 l l 0 2 6
coli
Citrobacter 15 5 6 2 8 36
freundii
Proteus spp 11 2 0 0 0 13
Pseudomonas spp 14 3 4 l 5 27 ------Total 136
Total number of aerobic isolates : 240 .
Anaerobic culture
Organism Gambia Zambia India Fiji PNG Total
Fusobacterium spp 18 3 7 l 17 46
Bacteroides spp 6 4 5 0 7 22
Anaerobic cocci 12 13 5 l 9 40
Propionobacterium sp 6 10 5 2 5 28 ------Total number of anaerobic isolates 136
148 Table 21 . No . of isolates per swab
Gambia Zambia India Fiji PNG Total
No growth 7 3 4 0 4 18
Single organism 10 l 5 2 0 18
2 organisms 15 7 0 l 9 32
3 16 7 6 0 15 44
>3 4 5 0 l 27 37
Table 22 . Analysis of single isolates
Single isolate No . of specimens
Non - lactose fermenting coliform bacilli 9
Anaerobic cocci 4
Coagulase negative staphylococci 3
Pseudomonas spp . 2
Total 18
Gram-negative aerobes such as E. cloacae , C. freundi i and
Proteus or Pseudomonas spp. were frequently isolated as shown in table 20 . E. coli was isolated less frequently (6/ 149 ) .
149 All the streptococci isolated were faecal streptococci, and were facultative anaerobes. Specimens from the Gambia were heavily contaminated with Proteus spp . The organisms were difficult to inhibit on the aerobic plates as they were resistant to nalidixic acid, gentamicin, tobramycin and were only sensitive to netilmicin at a minimum inhibitory concentration of 64 ug/ml. Pseudomonas spp . were often isolated from samples obtained in the Gambia.
Detailed speciation was not performed on these isolates.
Results of anaerobic culture
Fusobacteria were isolated most commonly from the swabs (table 20 ) .
Anaerobic cocci were also isolated frequently . Two or more anaerobes were recovered from the swabs on several occasions as shown in table
23. The fusobacteria were frequently isolated in association with
facultative anaerobes (42 samples,91%) . The frequency of isolation of all bacteria from the ulcers is listed in table 24.
150 Table 23 . Frequency of isolation of 2 or more anaerobes from the same sample
Anaerobes Number of samples
Fusobacterium and Bacteroides spp. 8
Fusobacterium and anaerobic cocci 18
Bacteroides and anaerobic coci 7
Bact . , Fusobac . , and an. cocci 4
Table 24 . Bacteria isolated from swabs
Organism ~~ total isolates
E. cloacae 14
C. freundii 10
Pseudomonas spp 7
Proteus spp 3
E. coli 2
S. aureus 1.6
CNS 14
Feacal streptococci 12
Fusobacterium spp. 12
Anaerobic coci 11
Bacteroides spp. 6
Propionobacterium spp . 8
151 Duration of ulcer and the presence of anaerobes
Anaerobes were only present in samples of less than six weeks
duration. Table 25 compares the duration of ulcers with recovery of
anaerobes.
Bacteriology of mud samples
The mud samples were cultured aerobically and anaerobically and the
results are shown in table 26. All samples had more than one isolate
present. All the samples contained anaerobic cocci and Bacteroides
spp. 3 contained fusobacteria, and 2 contained Clostridia spp,
whereas Clostidia spp. were never isolated from ulcers.
All the isolates recovered from the ulcers were stored at -70°C
until required.
152 Table 25. Duration of ulcers and recovery of anaerobes
Duration of ulcer No of ulcers
with anaerobes (weeks ) (%)
0- 6 54 (36 )
6-12 2 (l )
>12 0
Table 26. Number of isolates per sample of mud
No of isolates per sample Number Location
Single isolate 0
2 isolates 0
3 isolates 0
>3 isolates 4 PNG, India
153 Description of fusobacteria
Those Gram-negative, non-sporing rods which produced large amounts of n-butyric acid and no iso-butyric acid as end products of metabolism were regarded as members of the genus Fusobacterium.
All these isolates also converted threonine to propionic acid and thus fulfilled the requirement for membership of this genus. All the
Fusobacterium species were isolated from ulcers of less than six weeks duration.
The fusobacteria were recovered from the BHI plates, but were easily recognised on the BHI plates supplemented with rifampicin. The isolates were sensitive to metronidazole, penicillin and phosphamycin. They did not grow on Columbia blood agar plates incubated in air with 5% CO , nor on the MacConkey plates 2 and were thus obligate anaerobes. The two distinct morphological types isolated from 46 ulcers will be referred to as type 1 & type 2 fusobacteria.
Type 1 Fusobacteria
Colonial morphology
The 40 isolates that belonged to this group were identical. The organisms grew relatively quickly and colonies were visible at 24 hours but reached their maximal size by 36-48 hours. The optimal temperature for growth was 37°c. The colonies were 3-4mm in diameter, dull white in colour initially but became yellow with age.
They produced no haemolysis on blood agar nor any pigment. The edge of the colony was smooth and entire.
154 Colonies were dome shaped in appearance (fig. 18 ) . Growth on different media e . g . SHI, FABA or Wilkins-Chalgren agar produced no change in the rate of growth although the organisms showed considerable morphological pleomorphism. In broth culture the organisms grew readily and dispersed well throughout the medium.
They grew easily in BHI broth without blood supplementation. Broth cultures were malodourous due to the butyrate.
Cellular morphology
A Gram-stain showed long thin rods which displayed pleomorphism of
size and shape with some organisms short and almost caecal . The ends
were pointed. Others were filamentous and fusiform. The cells
stained evenly as Gram-negative, non-sporing rods ( figure 19 ) .
Type 2 fusobacteria
Colonial morphology
These colonies were smaller than type l colonies, about 2mm in
diameter . They were translucent, butyrous in consistency and grew
much slower than type l colonies. Type 2 colonies were not visible
at 24 but at 36 hours and attained their maximal size by 48-72
hours. They produced neither pigment nor haemolysis on blood agar .
The colonies were round with an entire edge and low convex shape
(Fig. 20 )
155 Cellular morphology
Gram stain showed marked pleomorphism of size and shape. The rods were larger than type l organisms and showed a central bulbous swelling. The edges were also pointed but the bacteria were very irregular in length . They stained irregularly but on the whole the organisms were Gram-negative ( figure 21 ) .
Guanine plus cytosine ratios
The guanine+ cytosine ratio was 29 . 2 mol % and 29 . 5 mol % for type l and 2 respectively.
156 Fig. 18. Type l fusobacteria colony
Fig. 19. Type l fusobacteria morphology ( xlOOO )
15 7 Fig. 20. Type 2 fusobacteria colony
Fig . 21 . Type 2 fusobacteria morphology (x lOOO )
158 Electron Microscopy of fusobacteria
Type l fusobacteria
The organisms showed significant morphological variation when grown on different media. Figure 22 shows these organisms harvested from supplemented BHI with 10% horse blood. The cells were 0.5-3u in diameter and 0.5-4.5u in length. The pleomorphism in shape was easily recognised. Some organisms were long and straight whereas others were curved. Figure 23 shows the same organism on Fastidious
Anaerobe agar with 10% horse blood. The pleomorphism is again evident, more so than on BHI. Figure 24 shows the organisms on
Wilkins - Chalgren agar supplemented with 10% horse blood. The picture shows again the considerable difference in morphology compared to the organism on BHI.
Type 2 fusobacteria
On supplemented BHI agar the pleomorphism was apparent. The bulbous swelling previously recognised on direct Gram stain was confirmed.
These organisms were larger than the Type l strains. The cells were
0.8-4.5u in length and 0.2u in width with a central width of 0.5u at the bulbous swelling ( figure 25 ) .
159 Fig . 22. Electron microscopy of type l fusobacteria (BHI ) ( x5000 )
Fig . 23 . Electron microscopy of type l fusobacteria ( FABA ) ( x5000 )
160 - Fig . 24 . Electron microscopy of type l fusobacteria ( WC ) (x5000 )
Fig . 25 . Electron microscopy of type 2 fusobacteria (BHI ) (x5000 )
161 Biochemistry of fusobacteria
The biochemistry profiles of the fusobacteria isolated are shown in table 27. All strains convert threonine to propionic acid which confirms their membership of the genus Fusobacterium. Both colonial types produced large amounts of gas in deep FABA after No H S was detected. All strains 24-36 hours incubation in air. 2 produced acid from glucose and type 2 strain fermented glucose and mannose. No acid was produced from fructose, lactose, sucrose, maltose, arabinose, raffinose or rhamnose. Indole, catalase, lecithinase, urease, lipase and oxidase were not produced. Aesculin and starch were not hydrolysed. Nitrate was reduced by all the strains. No digestion of gelatin or chopped meat occurred. These biochemical reactions differ from those species which the two morphological types of fusobacteria resemble namely, F.varium and
F. mortiferum. The differences are shown in table 28.
162 Table 27 . Biochemistry profiles of the fusobacteria
Test Type l Type 2
Catalase
Oxidase
Aesculin hydrolysis
Casein digestion
Hydrogen sulphide production
End products of acetic, butyric acetic,butyric metabolism & propionic acid propionic acid
Bile tolerance +
Carbohydrate fermentation Glucose Glucose & mannose
Nitrate reduction + +
Starch hydrolysis
D1 Aase production
Lipase
Conversion of threonine
to propionic acid + +
Indole
Gas production + +
163 Table 28. Differentiating characteristics of Fusobacterium species
Test F.mortiferum F.varium F.necrogenes F.ulcerans
Indole +
Aesculin + +
hydrlolysis
Nitrate + +
reduction
Gas producion + + + +
164 Antibiotic disc susceptibility tests
The fusobacteria isolated were sensitive to phosphamycin (300 ug / disc ) , kanamycin (1000 ugr/ disc ) and penicillin (2 u/ ml ) . All were resistant to rifampicin (7. 5 ug / disc ) .
Soluble cellular protein pattern of fusobacteria
Th e soluble cellular proteins were identical for all the isolates.
This is shown in figure 26. The reference standard ( Strep. faecalis ) is shown on the inner lane of the gels . The protein bands showed identical patterns for the two different morphological types of fusobacteria isolated . Compared with other Fusobacterium species, these protein patterns were completely different
(W.E.C.Moore, personal communication ) . That the Bacteroides and other Fusobacterium species differ is shown in figure 27 .
In view of the differences in the morphology , biochemical profiles and soluble cellular proteins, these fusobacteria have been identified as a new species and are referred to as Fusobacterium ulcerans . This nomenclature has been accepted by the International
Journal of Systematic Bacteriology . The type strain deposited with the National Collection of Type Cultures is NCTC 12111 and NCTC
12112 is a further reference strain.
165 Fig. 26. PAGE of fusobacteria isolates
166 s. faecalis F. ulcerans (l) F. ulcerans ( l) F. ul cerans ill F. ulcer ans ( 2) :E f-' · rt- ~ F. ulcerans (2) ::r N -..J B. 0 . loescheii rt- ::r B. (1) iJ intermedius "1 )::, C) U) rr1 F. nucleatum (l) 0 (1) 0 r) ...., F. nucleatum (2) f-' ...., °'-..J 1~·C F. nucleatum (1) U) 0 0 ...., CJ" F. nucleatum (l) OJ -.-, r) C rt- F. nucleatum (l) U) (1) "1 , 0 CJ" f-' • B. intermedius OJ OJ r) rt- f-' • (1) U) "1 0 f-' · f-' C OJ 3 rt- (1) U) Bacteroides spp.
Those gram negative rods which were non-sporing, anaerobic and which produced iso-valeric or valeric acids on gas liquid chromatography were tentatively regarded as belonging to the Bacteroides genus.
Their antibiotic sensitivities and biochemical profiles were recorded and are shown in table 29. The most frequently isolated species were B. thetaiotomicron and B. distasonis Both organisms have been well described previously and the strains isolated showed no difference from the recognised patterns. These
Bacteroides species were always isolated in association with other organisms.
Anaerobic cocci
Peptostreptococcus and Peptococcus spp. were isolated most commonly. One isolate of Veillonella was recovered.
Propionobacterium spp.
These bacteria were short Gram-positive rods which grew only
anaerobically and were sensitive to metronidazole. The organisms
produced large amounts of propionic acid.
Clostridium spp .
These thick Gram-positive spore-forming rods were only isolated from mud samples and not from ulcers. Further identification of these
bacteria was not performed.
168 Table 29 . Characteristics of Bacteroides spp isolated
Test Result
Oxidase
Catalase
Bile tolerance +
Antibiotic susceptibilities Sensitive to rifampicin
Resistant to kanamyc in
and vancomycin .
End products of acetic, isovaleric and metabolism valeric acids
169 Spirochaetes
Spirochaetes were visible on dark ground microscopy in 20% of the samples . Attempts to culture the spirochaetes were only partially successful. Of all the media used only NOS agar yielded growth of spirochaetes from three samples . The colonies were 4-5 mm in
diameter . They were white, fluffy and appeared round in the tubes .
The organisms were visible after one week's growth . However the
culture was mixed and further characterisation of the organism was
not possible. A carbol fuchsin stain shows the organism in mixed
culture ( fig 28 ) .
170 Fig . 28 . Spirochaete morphology (xlOOO )
171 Pathology
Biopsies were available on 20 patients from four endemic areas namely Gambia, Zambia, Papua New Guinea and India. The duration and treatment details were noted and are listed in table 30. In 17 patients the duration of the ulcer was known and of these, nine had been present for less than one month . The rest were between four and twelve weeks old. Seven patients had been treated with antibiotics
(penicillin= 6, metronidazole = 1 ) .
Light microscopic findings
l ) Epidermal changes
A) Haematoxylin and Eosin stain
The main abnormality seen in all patients was acanthosis around the ulcer rim accompanied by a variable degree of hyperkeratosis. The ulceration extended through the epidermis to the dermis. Around the ulceration the epidermis showed pseudoepitheliomatous hyperplasia.
Spongiosis was seen in the overlying epidermis of five biopsies all of which were less than six weeks in duration ( fig . 29 ) . A diffuse infiltrate of polymorphonuclear leukocytes in all layers of the epidermis accompanied the spongiosis. At the edge of the ulcer the epidermis appeared orderly and intact . In older lesions elongation of the rete ridges accompanied marked epidermal thickening. Remnants of stratum corneum were occasionally seen in the slough covering the ulcer.
172 Table 30. Duration of ulcers before biopsies
Duration (weeks ) Number of biopsies
0-4 9
4-8 4
8-12 4
unknown 3
173 B) Dieterle stain
Bacteria were seen in the epidermis in six patients, only one of whom had received antibiotics. All of these were ulcers less than one month old. In four cases the organisms were seen in the outer stratum corneum together with polymorphonyclear leukocytes. In two patients bacteria were seen scattered throughout all the epidermal
layers. For the most part these were pleomorphic Gram-negative rods
or Gram-positive cocci. The Dieterle stain proved very useful for
demonstrating all bacteria clearly. No fungi were identified in any
of these sections.
2) Subepidermal changes
A) Haematoxylin and Eosin stain
The pattern depended on the zone examined and the duration of the
ulcers and the treatment received. The ulcer beds were unremarkable
and showed granulation tissue with necrotic slough,
polymorphonuclear leukocytes and nuclear debris. At the base of the
ulcer vascular proliferation was common. None of the ulcers showed
extension to the subcutaneous tissues. Under the intact epidermis
there was marked oedema of the dermis particularly in ulcers less
than one month old. This was absent in patients with ulcers of short
duration who had received antibiotics (penicillin ) . The cellular
infiltrate was dense and ma ximal around the epithelial defect. Most
of the cells were polymorphonuclear leukocytes although in two
patients large numbers of eosinophils were seen. Lymphocytes were
also present in moderate numbers. Collagen bundles in the area of
the infiltrate were widely separated suggestive of oedema.
174 Next to the ulcers there was a dense inflammatory infiltrate of
chronic inflammatory cells around the blood vessels and adnexal
structures . Involvement of nerves was not apparent . Pigmentary
incontinence was seen in all biopsies and usually extended well
beyond the borders of the ulcer.
While the dermal blood vessels were dilated and increased in number
( fig. 30 ) there was marked thickening of the vessel wall with
endothelial proliferation in several chronic ulcers . There was no
evidence of vasculitis at any stage but one chronic ulcer showed
endarteritis obliterans.
B) Special stains
In acute lesions the sides and base of the ulcer were occupied by
inflammatory slough containing bacteria. In untreated lesions these
appeared to consist of a plug of necrotic material and organisms.
The predominant bacteria were rod-like or pleomorphic and were
Gram-negative . Long and arcuate forms were also seen (fig . 31 ) .
These differed from those described by Kuberski and Kotega (1980 ) .
In four untreated cases spiral bacteria were also found in the slough or at the ulcer rim ( fig . 32 ) . These were associated with other organisms. Gram-positive bacteria were rarely seen . The PAS stain failed to show any fungi and the Giemsa stain was useful for detecting the Leishman-Donovan bodies in those patients with leishmaniasis .
175 Fig . 29 . Pathology of tropical ulcer showing spongiosis
(haematoxylin and eosin ) ( x400 )
Fig. 30. Pathology of tropical ulcer showing vessel proliferation
(haematoxylin and eosin ) ( x400 )
176 Fig. 31. Dieterle stain showing bacteria (xlOOO )
177 Fig. 32 . Dieterle stain showing spirochaetes ( xlDDD )
178 Electron microscopy of tropical ulcer
Sections from seven patients were examined. The general features confirmed the light microscopic findings. In four acute cases the epidermal oedema was severe and the presence of polymorphonuclear leukocytes infiltrating the dermis was confirmed.
The predominating bacteria were rod shaped, round or elongated with cytoplasm containing ribosomes and a thin cell wall. The latter tended to be irregular (fig . 33 ) The appearances accorded most with the fusobacterial isolates from culture . By contrast organisms with the appearance of Bacteroides species examined in-vitro were only identified in two sections . These were rounder and had a more compact cytoplasm with thicker, more regular cell walls . The coliform organisms were not seen in the tissues sections. Small cocci were seen with dense staining cytoplasm and these were grouped around keratinocytes in some sections.
In two biopsies spirochaetes were observed in the deeper layers of the dermis and they were not associated with the other bacteria
( fig. 34 ) . These organisms showed sinusoidal periodicity and had three flagellae. Details of the insertion of the latter could not be adequately visualised in the sections. However these characteristics corresponded with those of spirochaetes (Hovind-Hougen 1976 ) . Some pleomorphic bacteria were present in macrophages or polymorphonuclear leukocytes. From their appearance it seemed that they had resisted intracellular digestion. Many disrupted bundles of collagen were seen in the vicinity of the clusters of bacteria.
179 Those patients with leishmaniasis and ulceration showed definite changes of leishmaniasis in the histological sections as shown in figure 16 . Several Leishman-Donovan bodies, together with a very mixed inflammatory cell infiltrate occupied the mid dermis. The inflammatory infiltrate consisted of lymphocytes and histiocytes mainly . In 2 sections, giant cells were seen. The overlying epidermis did not show any spongiosis.
Immunohistochemical studies
The skin biopsies of patients with tropical ulcers were studied by immunohistochemical markers to assess the inflammatory response in the dermis . The lymphocytic infiltrate in the dermis consisted of an equal number of T-cells and 8-cells as shown in figures 35 and 36 .
The lymphocytic infiltrate was concentrated mostly at the periphery of the ulcer.
180 Fig . 33 . Fusobacterium in the dermis (xlOOOO )
Fig . 34 . Electron microscopy of treponemes in the dermis (xlOOOO )
181 Fig . 35 . T-cells in the dermis (xlOO )
182 Fig. 36. B-cells in the dermis ( xlOO )
183 Patient serology
The sera of 20 patients and 10 controls were tested to a whole cell preparation of fusobacteria antigen. In 8 sera (5 patients and 3 controls ) there was bright fluorescence to the fusobacteria antigen visible (++ ) at a screening dilution of 1:16. Further dilution of
these sera to cover a range of titres from 1:16 to 1:1024 failed to produce fluorescence at the highe r dilutions . In two samples even
the original fluorescence could not be reproduced. Further characterisation of the antibody was therefore not carried out. The
results are shown in table 31.
Antibody production in patients and controls
The ELISA test showed that the patients and controls had very low
levels of antibody to most of the isolates from the ulcers. The
results are shown in table 32.
Treponemal serology
Sera of 20 patients and 10 controls were tested. All the sera showed a negative reaction to the test at a dilution of 1:4.
184 Table 31. Antibody production to fusobacteria.
Sample number Antibody production (fluorescence )
Dilution of serum tested
1:16 1:64 1:128 1:256
Patient 1 ++ + / -
2 ++ +/ -
3 +++
4 +
5 ++ +/ -
6 +
7 ++ + / -
8 +
Control 1 +++ +/ -
2 ++
3 ++ +
185 Rabbit serology
A whole cell preparation of Fusobacterium was used as the antigen.
The peroxidase labelled anti-rabbit IgG gave better readings at the
1:10,000 compared with the 1:5000 dilution. When the amount of IgG
produced by the rabbit was compared with normal rabbit serum which
served as the control only a small amount of antibody was produced.
This amount was much lower than the positive control (against
P.boydii ) . The result is shown in table 33.
When the rabbit serum was tested for production of IgM, there wa s a
small amount of IgM produced as is shown in table 34. As the amounts
of antibody produced were small the rabbit serum was not used to
detect the presence of fusobacteria in skin sections. The results of
the ELISA test showed that only small amounts of antibody were
produced in the rabbit in response to the repeated inoculations with
fusobacterium.
186 UNDER
UNDER
UNDER
UNDER UNDER
UNDER
UNDER
UNDER
Buffer
10
-.065
-.092
-.057 UNDER
UNDER 0.006
-.021
UNDER UNDER
UNDER UNDER
UNDER UNDER UNDER
UNDER
s
UNDER -.090
UNDER
UNDER UNDER
UNDER UNDER
UNDER
UNDER
ulcer
0.166
0.263
0.240
-.026
UNDER -.010 0.031
from
5
0.299
0.085
0.020
0.091
0.102
0.18
olated
s
i
Number
5 6 7 8 8
0.308
0.327 0.205 0.162
0.204
0.069
0.121
0.053
Patient
4
bacteria
-.098
0.078 -.024
UNDER
0.000
to
3
0.071 UNDER 0.077 0.074
0.039 0.027 0.234 0.249
0.076 0.018 0.074
-.061
0.140
ELISA
32
2
-.061 UNDER
-.040
-.041
-.002
UNDER UNDER -.096
-.094
)
Table
l
UNDER
-.021 UNDER
-.038
UNDER
UNDER
Serum
NHS
UNDER
UNDER 0.000 UNDER
UNDER
UNDER UNDER
UNDER
UNDER UNDER
Human
&
Normal
1:40
1:10
1:20
1:80
1:10
1:40
1:20
1:80
dilution
Antigen
Fusobacteria
Bacteroides
(NHS=
-..J f-'
CD
UNDER UNDER
UNDER UNDER
UNDER UNDER
UNDER UNDER
UNDER UNDER
UNDER UNDER
UNDER UNDER
UNDER UNDER
Buffer Buffer
014 014
080 080
102 102
.
.
.014 .014
.
10 10
-
-.042 -.042
-
UNDER UNDER
-
-
0.013 0.013
UNDER UNDER
204 204
054 054
077 077
054 054
072 072
8 8
. .
.
.
.
.
.
-
-.017 -.017
-
0
UNDER UNDER
-.074 -.074
0
0
s s
r
e
242 242
274 274
167 167
193 193
033 033
211 211
.
.
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.
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ulc
UNDER UNDER
0
0
0
0
0
0
0.062 0.062
from from
092 092
7 7 8
.
0.127 0.127
0.218 0.218
-.068 -.068
0.066 0.066
0
UNDER UNDER
-.008 -.008
231 231
148 148
023 023
.
.
.
isolated isolated
0.273 0.273
0
0
0.215 0.215
0.200 0.200
0.192 0.192 0.058
0.143 0.143
-
Number Number
243 243
278 278
246 246 158 158
058 058
255 255
269 269
5 5 6
.
.
.
.
.
.
.
0.199 0.199
0
0
0
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bacteria bacteria
to to
162 162
154 154 071 071
115 115
246 246
155 155
098 098
Patient Patient
4 4
.
.
.
.
.
.
.
0
0
0.258 0.258
0
0
0
0
0
ELISA ELISA
311 311
174 174
188 188
.
.
.
UNDER UNDER
0
0.241 0.241
0.225 0.225
UNDER UNDER
0
0.177 0.177
0
192 192 249 249
156 156
121 121
129 129
2 2 3
.
.
.
.
.
0
0
0
0.185 0.185
0
0.157 0.157
0.077 0.077
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continued. continued.
) )
32 32
022 022
047 047
024 024
075 075
l l
063 063
088 088
.
.
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.
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-.088 -.088 0
0
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0.064 0.064
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Serum
Table Table
069 069
000 000
.
.
NHS NHS
Human Human UNDER UNDER
UNDER UNDER
UNDER UNDER
UNDER UNDER
-.094 -.094 UNDER UNDER
-
0
& &
Normal Normal
forms forms
10 10
20 20
40 40
80 80
40 40
10 10
:
:
:
:
:
:
1
1:20 1:20
1:80 1:80
1
1
l l
1
1
NHS= NHS=
StreQ_tococcal StreQ_tococcal
dilution dilution
Coli Coli
Antigen Antigen
(
CD CD
CD CD I-' I-' Table 33 ELISA - Rabbit IgG Levels
Antirabbit IgG tested at 1:5000
Fusobacterium antigen 2ug/ 100ul buffer P.boydii 2ug/ 100ul <------> <------> Antibody Buffer Rabbit test serum Normal rabbit serum Buffer RTS P.boydii antiserum dilution <------> <------> <------> 1:10 0.849 1.388 1.425 1.329 1.435 0.961 0.691 0.240 0.563 1.420 l. 722 1.464 1:20 1.094 1.476 1.498 1.377 1.120 0.824 0.497 0.258 0. 792 1.497 1.650 1.450
1--' 1:40 0.554 0.073 0.669 1.411 1.604 CD 0.907 1.439 1.356 1.421 0.870 0.317 l. 350 '° 1:80 0.924 1.522 1.392 l. 353 0.574 0.483 0.236 0.035 0.522 1.464 1.451 l. 511 1:160 0.301 1.361 1.389 1.678 0.405 0.407 0.190 0.051 0.402 1.373 1.536 1.369
1:320 0.406 l. 312 1.310 1.608 0.463 0.414 0.149 0.107 0.251 1.308 1.522 1.381 1:640 0.349 1.354 1.380 1.411 0.229 0.248 0.127 0.043 0.199 1.269 1.427 1.348 1:1280 0.229 l. 329 l. 293 l. 235 0.184 0.208 0.108 0.036 0.030 1.235 1.408 l. 276
(RTS = Rabbit test serum) > >
0.042
0.210 0.172 0.019
0.121
0.079
0.272 0.034
antiserum
0.032
0.193 0.013
0.151 0.008
0.249
2ug/100ul
------
< 0.102 0.102 0.060 0.061
0.028
0.188 0.011
0.142 0.009 P.boydii
P.boydii
------
<
-.005 -.004
-.005 0.007 0.259 -.005
-.004 Buffer
>
-.006
-.005
-.003 -.005
-.005 -.004 -.005 0.000
Buffer
>
Levels
1:5000
IgM
at -.003
serum -.004 0.029 0.004
0.001
-.004 -.006 -.006
buffer
tested
rabbit
Rabbit
-.004
0.028 -.005 -.005
-.004 -.004
-.004
100ul
/ -
IgM
2ug
------
Normal
-.003
-.005
-.005 -.005 0.023 -.005
-.003 0.001 0.011
ELISA
> <
34
antigen
0.363 0.004 0.002
0.314
0.190
0.402 0.141
0.066
0.096
serum Antirabbit
Table
test
OVER
0.372
0.148
0.073 0.324
0.201
0.101
Fusobacterium
Rabbit
------
<
0.371
0.314
0.274 0.266 0.258
0.193
0.161
0.081
0.097
------
<
-.004
-.004
-.003 0.000 0.000 0.394
-.003 -.003
-.001
Buffer
:10
1:40
l 1:160
1:20
1:80
1:320
1:640
1:1280
Antibody
dilution
I-'
'°
D "echanisms of infection
Cytotoxicity
Cytotoxicity was demonstrated by morphological changes in the cell monolayers. The affected cells showed long filamentous tendrils
where damage was mild and rounding up of cells occurred when damage was more extensive . The nuclear-cytoplasmic ratio was 2: 1 before
exposure to the s upernatants (fig . 37 ) and 1 : 1 after cytotoxicity .
The changes associated with the cytotoxicity were observed 24 hours
after incubation . The rounded cells detached into the medium (figure
38 ) . Of all the cell lines tested, Vero cells were affected most
severely . The human embryonic intestine ( Int 407 ) cell line showed
similar but less severe changes . Th e percentage of Vero cells
affected by culture filtrates of t he test species is shown in t able
35 . The other cell lines namely, chimp liver, Chinese hamster ovary
and MRC 5, were not affected . No similar cytotoxicity occurred when
Vero cells were exposed to the uninoculated brain heart i nfusion
broth, Bacteroides supernatants or the coliform supernatants . The growth medium had no effect on the cells . The results of the
cytotoxicity tests using the cell lines other than Vero cells are
shown in table 36 .
Heating the s upernatants to 55°C or B0°C had no additional effect on the cytotoxicity . The degree of cytotoxicity was the same
as prior to heating.
191 Fig . 37. Vero cells with growth medium
192 ~ 38.. Vero cells showing cytotoxicity
193 Dialysis of the supernatants against PBS resulted in loss of cytotoxicity. As the supernatants were produced under anaerobic conditions, anaerobiasis was thought to be important for the persistence of cytotoxicity. The supernatants were therefore
dialysed against PBS with cysteine but cytotoxicity was still lost.
Concentration of the supernatant against polyethylene glycol (PEG)
increased the amount of cytotoxicity. The results are shown in table
37. This effect was not due simply to the concentration of proteins
in the medium (BHI) as concentration of BHI against PEG failed to
produce a similar result.
Equivalent dilutions of butyrate to that in the culture filtrates
caused the same morphological effect on the Vero cells as the crude
culture filtrates. The percentage of Vero cells affected by the
dilutions of medium containing butyrate were the same as the
corresponding culture filtrates of the fusobacteria. Butyrate
concentrations of 30mM or more affected 100% of the Vero cells.
These results are shown in table 38. Culture filtrate dilutions of
strains of Bacteroides fragilis , Enterobacter cloacae or
Streptococcus faecalis had no morphological effect on the Vero
cells.
194 Table . 35 . Percentage of Vero cells affected after 24 hours
incubation with 1 : 8 dilution of test culture filtrates
Species Vero cells affected
F. ulcerans (NCTC 12111 ) 10m~
F. ulcerans (NCTC 12112 ) 10m~ 501 Bacteroides fragilis 10 501 Enterobacter cloacae 10 501 Ste{2tococcus faecalis 10
195 Table 36 . Cytotoxicity in various cell lines excluding Vero cells
Sample & Cell Line
dilution
Chimp Liver Hamster ovary MRC 5 INT 407
F. ulcerans
NCTC12lll
1 :4 + ++
1 : 8 +/ - +
1:16 +/ -
F. ulcerans
NCTC 12112
1 :4 + ++
1 : 8 +/ - +
1:16
B. thetaiotomicron
1:4
[ . cloacae
1 :4 +
1 : 8
S. faecalis
1:8
196 Table 37. Results of dialysis of supernatants
Sample Dilution 1:2 1 : 4 1:8 1 :16
++ + F. ulcerans NCTC 12111 +++ +++ + F. ulcerans NCTC 12112 +++ +++ ++
B. thetaiotomicron +/ -
E. cloacae +/ -
Fusobacterium supn +/ -
dialysed against PBS
Fusobacterium supn +++ +++ +++ ++
dialysed against PEG
Fusobacterium supn +/ - +/ -
dial. agnst. PBS+cysteine
BHI
BHI dial. agnst . PBS
BHI dial. agnst.PEG
F.ulcerans supn filtrates
1-10
11-20
21-30 +
31-40
197 Table 38. Percentage of Vero cells affected after 24 hours
incubation with varying concentrations of butyrate
Concentration in medium (M ) Cells affected (%)
0.625 100
0.500 75
0.375 50
198 Protein precipitation and gel filtration
caused After ammonium sulphate precipitation, the supernatants severe damage to the Vero cells when the cytotoxicity experiments were repeated . A similar effect was observed when the ammonium sulphate was used as a control . Several fractions showed an increase used in in the solute concentration . These individual fractions were changes the cell culture experiments . However none of these produced to similar to those of the supernatant . The fractions were thought
be too dilute so the gel filtration was repeated with supernatant again fluid concentrated against PEG . Although several fractions was showed an increase in solute concentration, no cytotoxicity
observed when these fractions were tested .
199 Animal studies
The potential for synergy between aerobic, facultative and anaerobic of bacteria was studied with intradermal injections of mixtures animal organisms isolated from tropical ulcers. Two experimental models were used to simulate the cutaneous ulceration of tropical by ulcer. Synergy between two groups of bacteria was determined with a titration of the aerobic bacterial strains in association The single sublethal dose of one of the other anaerobic bacteria. was formation of an abscess after intradermal injection of bacteria
strongly dependent on the dose used.
Experimental infection in mice
In the C57 black mouse, cutaneous necrosis occurred with inoculation 9 10 of F. ulcerans NCTC 12111 ( figure 39 ) at a dose of 0.5 x
org/ ml. The lesions became necrotic, crusted and the skin sloughed. all to Thi s was not reproducible as some mice showed no reactions at In the the same dose of inoculation. No mice developed abscesses. two other mouse strains no local reactions occurred. Mice from the after strains (Balb/ c HeA 2 and C x S 12 ) became lethargic two days the the injections. The third day after the injection of local fusobacteria they died although there was no evidence of any
cutaneous reaction. The mouse model was therefore abandoned.
200 Experimental infections in guinea pigs
The reactions to subcutaneous injections were recorded as; l) Erythema: the area was measured and recorded in centimetres,
2) Induration: the induration was measured and recorded as above,
3 ) Abscess formation: this occurred when pus formed and ruptured
through the skin.
Determination of the minimal abscess forming dose
The minimal abscess forming dose was determined for each bacterium
alone and in combinations of two or more bacteria. Only abscesses
with pus formation were regarded as positive. The lowest dose
producing an abscess in at least 50% of the injections was regarded
as the minimal abscess forming dose (Brook and Walker 1983 ) .
The results of the injections of each of the bacteria are shown in
table 39. Of the anaerobic bacteria only B. thetaiotomicron 9 produced an abscess with an abscess forming dose of lxlO . F.
ulcerans failed to produce an abscess after intradermal injection 10 despite a high inoculum (l x 10 ) . At most erythema developed at
the site of inoculation . The aerobic coliform bacterium E. cloacae 10 produced an abscess with an abscess forming dose of l x 10 .
201 of single bacteria Table 39 . Reactions to intradermal injections
Organism injected Dose Result
7 E. cloacae l X 10 no reaction 8 l X 10 erythema 9 l X 10 erythema + induration 10 l X 10 abscess
107 B. thetaiotomicron l X erythema 8 l X 10 erythema + induration 9 l X 10 abscess 10 l X 10 abscess
107 F . ulcerans NCTC12lll l X no reaction 8 l X 10 no reaction 9 l X 10 no reaction 10 l X 10 no reaction
l X 1012 erythema
7 s. faecalis l X 10 no reaction 8 l X 10 no reaction 9 l X 10 slight erythema 10 l X 10 erythema
106 Anaerobic cocci l X erythema 7 l X 10 erythema 8 l X 10 erythema 9 l X 10 erythema
202 Inoculations with a combination of organisms
The results after inoculation of a combination of bacteria are shown
in table 40. The results can be expressed in groups according to their ability to cause subcutaneous abscesses in the guinea pig
model.
Group l induced abscesses when injected by themselves. These
included E. cloacae and B. fragilis
Group 2 did not induce abscesses when injected by themselves and
were never recovered from the abscesses when injected with other
organisms. This group included S. faecalis.
Group 3 did not produce abscesses when injected by themselves but
did survive and were associated with necrosis when injected with
other organisms in at least 70% or more of the abscesses. F.
ulcerans was included in this group.
Subcutaneous abscesses were formed after the inoculation of a
combination of the two anaerobic test organisms ( F. ulcerans and
B. thetaiotomicron ). Abscesses developed in 48 hours and reached a
diameter of 10-20 mm in 3-5 days . They drained in that time then
gradually healed. Injection of F.ulcerans and [.cloacae caused a
subcutaneous abscess with pus formation which drained spontaneously
through the skin after 2 days (fig . 40 ) . All those abscesses which
were not excised, drained freely and spontaneously healed over 6-7
days. The abscesses developed a superficial crust and gradually
healed from the outsides inwards. No guinea pigs died after the
inoculations.
203 The abscesses produced resembled those produced by E. cloacae 8 alone except that the dose used (1 X 10 ) was less than the dose 10 of the single organism (1 x 10 ) required to produce an abscess.
The inoculations were repeated on the back of the guinea pig and
produced similar results. The repeated inoculations were carried out
at different sites away from previous abscesses. Despite repeated
injections in the guinea pig model the clinical results were
similar .
Recovery of organisms inoculated
Aerobic and anaerobic cultures of swabs from the abscesses showed
the organisms originally inoculated in all cases .
Histology of experimental abscesses
The epidermis showed evidence of necrosis . Where early biopsies were
done just prior to rupture and the overlying epidermis showed
incipient rupture ( fig. 41 ) . Underneath the epidermis the dermis
showed oedema and dilated blood vessels . The inflammatory infiltrate
consisted mostly of polymorphonuclear leukocytes in the upper part
of the dermis. In the superficial dermis the organisms could be seen
on Gram stain. There was no evidence of vasculitis .
204 Fig. 39. Mouse model showing cutaneous necrosis
205 Fig . 40 . Guinea pig model showing abscess formation
Fig. 41 . H istology of experimental abscess
(haematoxylin and eosin ) (x400 )
206 Table 40. Inoculations with a combination of organisms
Key: E = Erythema (cm) I= Induration (cm ) A= Abscess
9 10 S. faecalis lxlO + F. ulcerans NCTC 12111 lxlO
Day 1 2 3 4 5 6
E,B E,7 E,5 E,l
I,3 I,l
9 10 E. cloacae lxl0 + F. ulcerans NCTC 12111 lxlo
Day 1 2 3 4 5 6
E,5 E,5 E,4 E,3 E,3 E,l
I,2 I, 4 A A A A
8 E. cloacae lxl0 + F. ulcerans NCTC 12111 lxlOlO
Day 1 2 3 4 5 6
E,4 E,3 E,2 E, .2 E,2 E,l
I,2 I,3 A A A A
9 10 Anaerobic cocci lxl0 + F. ulcerans NCTC 12111 lxlo
Da y 1 2 3 4 5 6
E,4 E,2 E,l
207 DISCUSSION
Although tropical ulcer is endemic in certain parts of the tropics it is not confined to t hese areas . Du ring this investigation, patients were studied in f ive tropical countries namely Zambia,
Gambia, India, Papua New Guinea and Fiji . The prevalence of the disease varied from country to country and also from one part of a country to another . For instance in the Gambia the disease was more frequently seen in the rural areas than in the capital, Banjul . In
Papua New Guinea ulcers were common in all areas studied . In Fiji the disease has been very common up to five years previously . Now few patients were developing ulcers, possibly a reflection of the impact of the Figian government's commitment to improvement of sanitation and living conditions in the rural areas . Nearly all villages have f l ush toilets and piped and regular visits from health inspectors ensure that toilets and water supplies are maintained .
The seasonal variation in the incidence of ulcers was also confirmed during this study particularly in Gambia and Zambia . The study in
Fiji took place after the rainy season which could have accounted for the few cases observed . H owever this is unlikely as health workers in the area, who were well versed with the features of tropical ulcer, reported t hat the numbers of patients with ulcers were definitely decreasing . In Papua New Guinea (PNG ) where the
rainfall was similar throughout the year, ulcers occurred with equal
frequency irrespective of the season .
208 The disease frequently affected children between the ages of 5 and
15 years although it was also seen in older patients. The disease
was uncommon in patients over the age of 45 years. In Gambia and
Papua New Guinea where more patients were over the age of 15 years,
this increase in age could be attributed to the number of adult
women who worked as farmers (in Gambia ) and in fishing (in PNG ) and
who were thus exposed to the risk of disease while wading through
the water to catch fish by line or trap nets (Robinson et al 1987 ) .
While most cases were seen in rural communities, the patients in
southern India were town dwellers. The clinical manifestations of
the disease were similar in all the areas studied and most ulcers
occurred on the lower legs. The same pattern of evolution was
identified in all the areas. Only one patient developed a squamous
cell carcinoma. The clinical manifestations were thus confirmed in
all areas studied (Adriaans 1988 ) .
The nutritional status of the patients was assessed objectively with
three indices to determine whether or not tropical ulcers were
associated with malnutrition. The results showed that nutritional
status did not affect the onset of tropical ulcers. Several
populations of children and adults were studied in different areas
and poor nutritional status never correlated with the onset of
tropical ulcers. Overt clinical malnutrition was rarely observed in
these patients though man y looked clinically malnourished, although
not grossly so. Dietary histories did not suggest a critical
shortage of first class protein. The majority of patients could
recall eating meat or fish in the preceeding week. Although fresh
fish wa s less abundant in the rainy seasons, dried fish was freely
available in all areas.
209 Ankle oedema was rare and when present was confined to the leg affected with the ulcer. The area of oedema was usually close to the confines of the ulcer. The haemoglobin levels in the 64 patients where this measurement was done were always above 10 G% even though
many were clinically suspected of anaemia. Only one patient in the
Gambia had a sickle cell trait with a normal haemoglobin. In this
patient the ulcer was on the middle third of the leg and on the
lateral border. Clinically this was more in keeping with a tropical
ulcer than a sickle cell ulcer. There wa s no difference in the
nutritional status of the ulcer patients and an age and sex matched
control population in the same area although malnutrition was common
in both groups studied. These findings suggest that malnutrition per
se is not a major contributory factor in the aetiology of tropical
ulcer.
The aetiology of tropical ulcer is obscure. While it is regarded by
some workers as a non-specific response to injury, there is evidence
that the disease is transmissible. McAdam (1966 ) for instance was
able to induce tropical ulcers in normal healthy subjects by
inoculating pus from an affected individual into normal skin. This
suggested that infection played a role in the pathogenesis. Most
previous reports on the bacteriology of ulcers have concentrated on
aerobic cultures although several workers reported the presence of
fusiform bacilli in smears. These had not been fully characterised.
Despite this, many authors have alluded to the importance of these
bacteria in the aetiology of tropical ulcers.
210 In this study the ulcers were investigated bacteriologically for both aerobic and anaerobic organisms. The successful isolation of anaerobic bacteria depended largely on obtaining good samples and then maintaining them under strict anaerobic conditions until the
final analysis was done. Because the patients were seen a long way
from the investigating centre, the swabs for bacteriological assessment had to be transported anaerobically until they were cultured. A suitable new system for transporting the samples was
devised (Adriaans et al 1986 ) . The Hungate tubes used for this
purpose served as reliable individual culture systems and kept the
samples in an anaerobic environment. The anaerobic organisms,
including the fusobacteria which are believed to be important in the
pathogenesis of this condition, remained viable under thse
conditions. The advantages of these tubes included the following:
1) they were reliable and maintained anaerobiasis
2) they were easy to use
3) they were convenient for storage
The weight of the tubes was their only disadvantage. Two tubes were
used per patient. Each contained 10 ml of either transport fluid.
The glass tubes were fragile, another potential disadvantage as the
tubes always had to be transported by hand luggage throughout the
field trips. However the advantages clearly outweighed the
disadvantages.
211 In this study several anaerobic bacteria were isolated (Adriaans et al 1987a) . Their successful isolation was possible because:
1 ) a reliable transport medium was used.
2) current techniques facilitated the isolation of anaerobic
bacteria. These fastidious organisms could be recovered from
the specimens with relative ease.
3) isolations were performed in an anaerobic cabinet
4 ) a number of selective media were used.
This study confirmed the presence of anaerobes in these wounds. The
most frequently isolated anaerobic microorganisms were
Fusobacterium species, Bacteroides species and various anaerobic
cocci. Fusobacteria were isolated only from ulcers less than six
week's old and none of the patients from whom these organisms were
recovered had received any antibiotics.
From this study a previously unreported species of Fusobacterium
was identified. It differed in its morphology, biochemical reactions
and in its soluble cellular protein pattern from other previously
described species of Fusobacterium
This new species was isolated from several ulcers (Adriaans and
Drasar 1987 ) and has been named Fusobacterium ulcerans in keeping
with it's source of isolation (Adriaans and Shah 1988 ) . The organism
has been deposited with the National Collection of Type Cultures and
is identified as NCTC 12111. This strain represents those bacteria
described as type l fusobacteria which were the most frequently
isolated species. A further representative strain has been deposited
as NCTC 12112. This corresponds with type 2 fusobacteria.
212 No other species of Fusobacterium were recovered . Group l fusobacteria formed t he majority and totalled 40 isolates . These isolates resembled Fusobacterium varium most closely but there were distinct biochemical differences . The second group of fusobacteria resembled F. mortiferum most closely. Yet they too showed several biochemical differences . Although the fusobacteria isolated in this study showed morphological and biochemical variations, the soluble cellular proteins of all the isolates were identical . Thus, despite interspecies variation, these organisms probably all belong to the same species.
The fusobacteria were isolated from swabs taken from the bases of ulcers . However the electron microscopic studies confirmed their presence in the dermis of the skin biopsies . These organisms therefore not only colonise the surface of the ulcers but also invade the dermis . The exact mechanism of invasion is not clear.
However it seems likely that invasion follows a breach in the surface of the skin which allows these organisms to enter. The skin
is a good barrier and extremely effective at keeping organisms out.
The histories of trauma lend support the hypothesis that trauma is
required to provide the portal of entry for these organisms. Those
patients treated with antibiotics early in the disease recovered
well and the ulcers healed easily . The fusobacteria isolated were
sensitive to penicillin and metronidazole . The improvement after
administration of either of these antibiotics may be related to
removal of these organisms . This also supports the hypothesis that
infection is important in the aetiology of tropical ulcer .
213 The source of these organisms was also investigated. Identical organisms were isolated from mud taken from the areas where patients with tropical ulcers lived. Whether these are human or animal derived bacteria is unknown. It is surprising that these organisms have not previously been documented if they were of human origin. In all areas studied animals roamed freely, sanitation was poor, stagnant water wa s commonly observed and it is likely that these bacteria are of animal origin.
The presence of anaerobes, particularly fusobacteria and spirochaetes , has been reported from several conditions such as cancrum oris , hospital gangrene and foot rot in sheep. The latter is a condition not too dissimilar from tropical ulcer. A necrotic lesion develops on the foot in wet or humid conditions.
Fusobacterium necrophorum has been implicated in the aetiology of this disease (Hine 1984 ) . F. necrophorum is known to be toxic to the epidermis in rats and has been shown to be toxic to Vero cells in culture (Grenier et al 1985 ) . Likewise Treponema dysenterica is cytopathic to intestinal cells in combination with other bacteria
(Lysons et al , 1978 ) . It is thus likely that the combination of organisms is critical for the development of such diseases and synergy between anaerobes and aerobes may be mechanism whereby these organisms produce disease.
Fusobacteria are normall y found in the faeces but they have been
isolated from the gingiva in patients with gingival and periodontal
disease (Moore et al 1984 ) . Fusobacteria are also thought to play
a role in cancrum oris, a mixed infection seen in malnourished
children (Finegold 1977 ) . In cancrum oris, extensive ulceration of
the skin occurs predominantly around the mouth.
214 The tissue necrosis develops rapidly, in much the same way as tropical ulcer. However in cancrum oris the affected individuals are severly malnourished or have some underlying condition such as lymphoma. The fusobacteria involved in cancrum oris have not yet been characterised.
The pathogenicity of the fusobacteria isolated from the ulcers was tested in an in-vitro cell culture system. Of all the isolates tested only fusobacteria caused this effect. The significance of this finding may be interpreted in the light of the sudden breakdown of tissues at the onset of the ulcers. Damage to the cells ma y be due to toxic concentrations of butyrate which have previously been shown to be of aetiological significance in periodontal disease
(Touw et al 1982). The fusobacteria do not produce haemolysin, nor do they digest casein which may contibute to the pathogenesis of the infection.
The histology showed that tropical ulcer was associated with large numbers of unusual bacteria. The predominant organisms in acute untreated lesions corresponded most closely to Fusobacterium ulcerans and some Bacteroides spp. Gram-positive bacteria were scantily distributed at the sites of tissue destruction. The ultrastructural appearances of spirochaetes were likely to be those of the Treponema spp. becaus e of their flagellar pattern, length and periodicity . Healing wa s associated with acanthosis and loss of dermal oedema and the absence of bacteria was associated with antibiotic treatment. These features differed from those in Buruli ulcers where the disease affects the subcutaneous tissue more extensively and epidermal proliferation does not occur to the same extent as in tropical ulcer.
215 No gram-positive cocci nor crusting were seen as in ecthyma .
From the material examined, there was no evidence to implicate vasculitis or a thrombotic process in the pathogenesis of tropical ulcers . All the vascular changes were likely to have resulted from the healing process and were typical of those seen in other chronic ulcers (Adriaans et al 1987b ) . No spirochaetes were identified in arterioles or arteries which suggested that spirochaete associated endarteritis was not a factor . The destruction of collagen, the disruption of fibroblasts and inflammatory cells in the vicinity of large numbers of invading bacteria, suggest that the bacteria are cytotoxic . This was confirmed by the in-vitro cytotoxicity studies which showed that Fusobacterium ulcerans , but not Bacteroides spp . isolated from the ulcers, is cytotoxic to Vero cells in culture . Immunohistochemical stains showed a number of 8-cells in the inflammatory infiltrate which suggests that the fusobacteria could stimulate the production of antibodies locally .
Although anaerobes are frequently associated with infections, information on the host's response to infection, especially with regard to antibody production against these organisms is limited.
There are no previous reports on the host response to the infection in tropical ulcer . Various methods have been used to assess antibody response . These include gel diffusion, passive haemagglutination and immunofluorescence to demonstrate the presence of antibodies to bacteria such as members of the Bacteroidaceae in patients with infections caused by Bacteroides or Fusobacterium The results of such investigations have frequently shown the presence of antibodies to these bacteria but the significance of these antibodies has not been clarified.
216 Sonnenwirth (1979) concluded that the increased titres of antibodies reported in association with infections were most often based on homologous isolates (the patient's own strains). This may reflect the strain and species specificity of antigens. With more sensitive tests (such as ELISA ) even low titres of antibody may be detected.
The sera from tropical ulcer patients were investigated for antibody production by an ELISA test. The same species of Fusobacterium was isolated from several ulcers which suggested it was of aetiological significance. The antigen was presumed to be constant and the antibody titre therefore comparable between patients. However no significant amount of antibody was produced in response to this infection. This may explain why patients develop recurrent infections over man y years . This is the first report of antibody responses in patients with tropical ulcers.
217 CONCLUSION
Tropical ulcer is a specific form of cutaneous infection which results from an infection with a mixture of organisms such as
Fusobacterium ulcerans , coliform bacilli and spirochaetes. The most likely method of infection follows cutaneous trauma. When there is a breach in the skin the bacteria enter and multiply. The presence of a combination of organisms favours their growth. It seems likely that the infection not merely a polymicrobial infection but rather a synergistic infection as has been shown by in-vivo synergy experiments. Once the bacteria enter the skin they multiply and cause damage locally in the skin. This study has also shown that a combination of organisms is necessary for the development of a tropical ulcer.
Further work is needed to characterise the spirochaetes. Improved methods which will allow the growth of the spirochaetes, should provide details on their biochemical and further ultrastructural properties. Once the spirochaetes are maintained in-vitro their full
role in the pathogenesis of tropical ulcer can be assessed.
Similarly the new species of Fusobacterium isolated should be
tested to see if in fact it stimulates the local antibody production
it the skin (Mangan et al 1983 ) . This would prove the hypothesis
that local antibody production prevents dissemination of disease.
Tropical ulcers heal easily when the patients are treated with
penicillin. The response is improved when treatment is started
early. This corresponds to the stage when there are many organisms
present as few are found in the late lesions.
218 This supports the hypothesis that infection is important and of the organisms isolated the fusobacteria are susceptible to penicillin.
The improvement may thus coincide with removal of those bacteria .
Although the disease has a low mortality it is very debilitating and
has a significant morbidity. With better sanitation, health care and
housing conditions this disease should be eradicated .
219 Appendix la. Tropical Ulcer Questionnaire
Guide to Questionnaire General points If the answer to the question is not known leave blank. Put in as much or supplementary information es you like under "additional comments" in each section of the questionnaire if appropriate. Where answer to question is Yes/No or Sex M/f ring e.g. Yes/No.
SECTION I e.g. [5) ~ if uncertain fill in box number of ege category es indicated means ege )0-)9 NOT 5 yeera old.
Household defined es those who normally eAt together i.e. two wives in separate huts count es two households. Exclude visitors: include children away et school.
Town: 2,000 inhabitants or more.
Animals be es precise es possible about what animals .ere kept where.
Hain activity describe e.g. desk, outdoor in fields etc.
SECTION II
To question "When did it start?" give interval from first medical contact if possible. lhis means orthodox medical contact e.g. community nurse, health centre, hospital NOT traditional or herbalist. family history of TPU if positive include simple family tree of first and second degree relatives if possible and arrow index case e.g. Try to establish whether the relative was in contact with the patient at the time the ulcer developed.
S[CTION II I
Past medical history - leave blank unless certain. Ignore acute inter current conditions e.g. measles, malaria. SECTION IV
See instructions on sheet. Under "other obvious scars" ignore tribal markings. SECTION V
Under distribution give approximate indication e.g. "anterior chest and abdomen" or "all over except face". Dental O.M.f: enter total number of decayed, missing or filled teeth.
220 Appendix la. continued
Tropical Ulcer Questionnaire Case [1] Control [2] [ Section 1: ENVIRONMENT NAME: •.•.•.••.•.••...•••••.. , . , , , , . , •• , . SEX: M/ F No •••••••••••••••
.DATE PATIENT FIRST SEEN (i.e. et health centre/ hospital) ......
AGE: (Age given) •••••••• Confirmed by medical or birth record YES/ NO
If uncertain, estimate:
[1] 0-4 yrs (2) 5-9 yrs [J] 10-19 yrs (4) 20-29 yrs [5] J0-39 yrs [6] 40 or over HOU SEIIOLD: (e) Number of adults [ (b) Number of children Exclude visitors, include children away et school HOME: [ (1] In town [2] In village [J] Isolated dwelling Number of houses within 50 yds [ . Name of nearest village/town ••.••.••••••••••••••••.••.••••••...••..••••••
Number of rooms ] Building materials: walls roof
Floor area ] sq yds
ANIMALS: What domestic animals ere kept in close proximity (e.g. pigs, dogs, include poultry):
Where kept? By day •..•••...•...•..•.••• Any animals kept inside home?
By night •••.•••..•.••••.• ; •
ACTIVITY:
Main activity, e.g. job/ school
If seasonal, give details
Activity/job of head of house
HAZARD: Possible predisposing factors to TPU, e.g. trauma, wading, splash- --- ing mud •••....•••.•••••..•••.•..••.•.....•...•..••..••.•...... •
HYGIENE: Washing facilities et home or after main activity - river, pond, well, piped water:
Does availability of water vary with season? ...... How many buckets of water used by family each day ..•...... Drying: custom, practices eg after washing ...... Is soap readily available YES/ NO Cost of bar of soap Has the information on this sheet been obtained by home visit YES/ NO
Additional comments to be made overleaf
221 Appendix la. continued
Section II: HISTORY Of ULCER
ULCER: How end when did it start?
TRAUMA: Preceding trauma Y[S/NO If yes, describe (including insect bite history)
PAIN: Painful IS/ WAS/NO
BLE[OING: Has it bled Y[S/NO
PAST HISTORY OF PREVIOUS ULCERS (dates if possible)
FAMILY HISTORY: Family history of TPU Y[S/ NO Relatives effected ••.•.•••••.•.•.••••••.••.....•.....•.. When ••.••••••••.••••...•••••••.•.•••.•..•. Was the affected relative living in the same household es the patient? Y[S/ NO Family tree - Complete if family history is positive
TREATMENT: Past treatment of present lesion Saliva applied? Y[S/ NO If yes, how ••••••.•.•••.•••••.
Traditional herbs YES/ NO Dressings YES/ NO Antibiotic details: Topical Systemic
Additional comments to be made overleaf
222 Appendix la. continued
Section Ill: MEDICAL/NUTRITIONAL
NUTRITION: STAPLE How often hes animal protein (fish or meet) been eaten in
the past week?
Does this very according to season? YES/ NO
Whet did you eat yesterday for (e) breakfast •••.•.••.•••••••••...•.•...• , . , , , , , , , , , . , , • , (b) lunch ...... (c) evening meal •••••••..•.••...•••..•••••••••••..•.....• (d) other times •••.••••••• •.•.•.. . , .•• ....•... , ...... , , ,
/ NO HEALING: Are cuts etc, slow to heel? YES
MEDICAL HISTORY: leprosy, ls there e definite history of past major medical condition, e.g.
Kwashiorkor, tuberculosis etc:
ADDITIONAL COMMENTS: rel~vRnt e.g. about staple/variations in diet, medical l1istor~, if thought
Continue overleaf if necessary
223 ~ndix la. continued
SECTION IV Map position of ulcer(s) on body diagram as an open circle, follow by number to indicate maximum diameter of ulcer in ems, e.g. 5 means ulcer of maximum diameter 5 ems.
Scars that you ere confident ere heeled ulcers should be similarly mapped es closed circles, e.g • 5. Indicate other obvious scars e.g. trauma, by lines to give approx. size
APPEARANCE:
Slough Edge Bleeding Surrounding oedema Tender Deep structure involvement Regional lymph nodes ?Malignant charge Smell lf there ere multiple ulcers end you wish to refer to one of them, label the ulcers by letters, e.g. b C) 5 on lhe diagram
Additional comments to be made overleaf
224 Appendix la. continued
Section V: General Examination
MEASURE: WEIGHT (to nearest 1/2 kg or lb) HEIGHT ( to nearest cm or 1/2")
to protection, CLOTHING: At time patient first seen with reference shoes, trousers, dress etc. If dress, how far down (knee, calf, ankle). Comment if clothing during main activity is different ......
DISTRIBUTION SKIN:
Ringworm YES/ NO Specifically Tinee imbricate YES/NO
Scabies YES/NO
Impetigo YES/ NO
DENTAL:
Teeth: D.M.F.
Gums (assessment of gingivitis) GOOO / POOR / BAD
Betel nut chewer YES/ NO
MEDICAL: Other major medical condition definitely present, e.g. leprosy, TB, obvious malnutrition, big spleen
Additional comments to be made overleaf
Form completed by: INVESTIGATIONS
et: village/health HG centre etc Serum date: Bacteriology: Gram stein, culture
Biopsy Photograph
225 Appendix lb . Peptone yeast glucose agar (transport fluid )
INGREDIENT AMOUNT (gr)
peptone 0 . 5
trypticase 0 . 5
cysteine 0 . 05
yeast extract 1.0
sodium sulphoxalate 0.03
rezazurin 0 . 4 ml
distilled water 100 . 00 ml
hemin solution 1.0 ml
vitamin K solution 0 . 02 ml
salt solution 4. 0 ml
Salt solution gr
CaC1 (anhydrous ) 0 . 2 2 MgS0 (anhydrous ) 0. 2 4 K HP0 1.0 2 4 1.0 KH/04 NaC0 10 . 0 3 NaCl 2. 0
226 Appendix le. Medium for spirochaete isolation.
NOS agar (Leschine et al 1980 ) .
INGREDIENT AMOUNT (gr)
Heart infusion broth l. 25
Trypticase 1.0
Yeast extract 0.25
Sodium thioglycollate 0.05
1-Cysteine HCL 0.1
1-Asparagine 0.025
Glucose 0.2
Noble agar 0.7
ml / 100 ml
Thiamine pyrophosphate 0.3
Volatile fatty acid 0.2
Normal rabbit serum 2.0
10% Sodium bicarbonate 2.0
227 Appendix ld. Ingredients for polyacrylamide gel electrophoresis
Resolving gel:
a ) Sterile distilled water 9 . 93 ml
b) Acrylamide/ bis 6 . 8 ml
c ) Resolving buffer 2. 125 ml
d) APS 10% 100 ul
e ) TEMED 10 ul
The first three ingredients were thoroughly
mixed after each addition.
Stacking gel :
a ) Sterile distilled water 18.3 ml
b) Acrylamide/ bis 2.7 ml
c ) Stacking buffer 3. 0 ml
d) APS 10~~ 50 ul
e ) TEMED 40 ul
f) Bromophenol blue dye ( BPB ) 7 drps
APS - Aqueous Ammonium persulphate solution
10% wt / vol. Freshly prepared
TEMED - N,N,N ' ,N' - tetramethylethylenediamine
BPB - 0 . 25% Bromophenol blue in water wt / vol
228 Appendix le . Medium used to support the growth of cells
l ) Medium 199 (Gibco, U. K. ) .
90 ml sterile distilled water
10 ml 199 medium
1-5 drops 2N NaOH ( to change the ph as indicated
by a colour change to orange yellow )
5 % fetal calf serum
0. 22% sodium bicarbonate
20mM Tricene pH 7. 6
antibiotic solution 0 . 5%
2) Dulbecco's Modified Eagles Medium (Flow Lab . U. K. )
10% fetal calf serum
20 mM tricene
0. 22% Sodium bicarbonate
25 ug / ml gentamicin
229 SUPPLIERS
Microscopes Zeiss PO Box 78
Welwyn Garden City
Herts
Leitz (cytotoxicity )
48 Park Street
Luton
Beds
Nikon
Richmond Road
Kingston- on-Thames
Surrey .
Homogeniser Gallenkamp Belton Road West
Loughborough
Leicestershire
Sonicator MSE Manor Royal
Crawley
Sussex
Multichannel pipette Jencons Scientific (Titertek ) Leighton Buzzard
Beds
230 Filters Millipore Harrow
Middlesex
Whatman
Denmark
Oxygen free gas 8 . 0. C. Special Gases Division
London
Culture media Oxoid Basingstoke
Hants
Difeo
East Molesey
Surrey
Lab. M
Sal ford
Cheshire
Gibco
Uxbridge
Middlesex
231 Dessicator Polysciences Ltd Moulton Park
Northampton
Northants
Hungate tubes Bellco Glass Vine lands
New Jersey
U.S.A.
Anaerobic cabinet, Don Whitley
Disposable loops & Shipley
Multichannel cannula Yorkshire
Electrophoresis tank Biorad Watford Business Park
Wat ford
Herts
Glass beads Ballotini Stanmore
Middlesex
Sephadex beads Pharmacia Chemicals
Hounslow
Middlesex
Anti-rabbit immunoglobulin Dako High Wycombe
Bucks
232 Antiseptic solution I . C.I. Pharmaceuticals Macclesfield
Cheshire
Anaesthetics Astra Pharmaceuticals Kings Langley
Bucks
Janssen Pharmaceuticals
Wantage
Oxfordshire
Biopsy punch Stiefel High Wycombe
Bucks
Scalpels Gillette Isleworth
Kent
Silk sutures Ethicon (Mersilk) McCarthy Supplies
Dagenham
Essex
Spiral plater Spiral Systems Clough Pike
Cinncinati
U. S.A .
233 Metronidazole discs May and Baker Dagenham
Kent
Streptex kit, Wellcome Diagnostics
Syfacard kit. Dagenham
Kent
Petri dishes Sterilin
Farnham
Hants
Microwell plates Flow Laboratories
Rickmansworth
Herts
API kits API Laboratories
Monralieu
France
Duolite resin BDH Chemicals
Poole
Hants
Chromatograph Philips and computer Cambridge
Cambs
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